Jonathan T.C. Liu

All Publications

• 3D pathology-guided microdissection. bioRxiv : the preprint server for biology Hsieh, H. C., Gao, G., Han, Q., Brenes, D., Baraznenok, E., Yan, R., Serafin, R., Bishop, K. W., Wang, R., Konnick, E. Q., Pritchard, C. C., Figiel, S., Hamdy, F. C., Mills, I. G., Reder, N. P., Reddi, D. M., Paulson, T. G., Grady, W. M., Valk, J. E., True, L. D., Haffner, M. C., Rao, S. R., Woodcock, D. J., Liu, J. T. 2025

  Abstract

  Traditional micro- and macro-dissection techniques enable the extraction of localized regions in thin tissue sections for molecular analysis. Despite the growing use of 3D microscopy, analogous methods for volumetric microdissection are lacking. We have developed a 3D microdissection method based on computer numerical controlled (CNC) milling integrated with open-top light-sheet microscopy. We demonstrate the ability to study tumor evolution along convoluted 3D branching architectures, which is inaccessible to 2D methods.

  View details for DOI 10.1101/2025.11.20.689586

  View details for PubMedID 41332752

  View details for PubMedCentralID PMC12667760

• Deep-learning triage of 3D pathology datasets for comprehensive and efficient pathologist assessments. bioRxiv : the preprint server for biology Gao, G., Yan, R., Song, A. H., Hsieh, H. C., Barner, L. A., Wang, F., Brenes, D., Chow, S. S., Wang, R., Bishop, K. W., Liu, Y., Farre, X., Divatia, M., Downes, M. R., Vakar-Lopez, F., Lal, P., Burke, W., Madabhushi, A., True, L. D., Reddi, D. M., Grady, W. M., Mahmood, F., Liu, J. T. 2025

  Abstract

  Standard-of-care slide-based 2D histopathology severely undersamples spatially heterogeneous tissue specimens, with each thin 2D section representing <1% of the entire tissue volume (in the case of a biopsy). Recent advances in non-destructive 3D pathology, such as open-top light-sheet microscopy (OTLS), enable comprehensive high-resolution imaging of large clinical specimens. While fully automated computational analyses of such 3D pathology datasets are being explored, a potential low-risk route for accelerated clinical adoption would be to continue to rely upon pathologists to provide final diagnoses. Since manual review of these massive and complex 3D datasets is infeasible for routine clinical practice, we present CARP3D, a deep learning triage framework that identifies high-risk 2D cross sections within large 3D pathology datasets to enable time-efficient pathologist evaluation. CARP3D assigns risk scores to all 2D levels within a tissue volume by leveraging context from a subset of neighboring depth levels, outperforming models in which predictions are based on isolated 2D levels. In two use cases - risk stratification based on prostate cancer biopsies and screening for dysplasia/cancer in endoscopic biopsies of Barrett's esophagus - AI-triaged 3D pathology, enabled by CARP3D, demonstrates the potential to improve the detection of high-risk diseases in comparison to slide-based 2D histopathology while optimizing pathologist workloads.

  View details for DOI 10.1101/2025.07.20.665804

  View details for PubMedID 40777412

  View details for PubMedCentralID PMC12330501

• Delivery of a fibrin-binding hemostatic polymer ameliorates neurovascular damage and neural tissue loss after traumatic brain injury. Science advances Han, Q., Brenes, D., Bishop, K. W., Pichon, T. J., Ling, M., McPheron, G. D., White, N. J., Pun, S. H., Liu, J. T., Sellers, D. L. 2025; 11 (29): eadw7425

  Abstract

  Traumatic brain injury (TBI) often induces blood leakage into brain tissues, which causes further tissue loss after the initial injury. To mitigate this secondary injury, we hypothesized that delivery of a fibrin-binding hemostatic polymer, PolySTAT, would act as a molecular patch and ameliorate brain vessel damage following TBI. We developed a three-dimensional (3D) pathology and analysis workflow to quantify the effects of PolySTAT versus a control polymer, PolySCRM, on neurovascular networks and neural tissue in whole mouse brains. Using a panel of fluorescent probes, our 3D pathology pipeline revealed that PolySTAT treatment preserves neurovascular density and function, reduces hypoxia and blood extravasation, and reduces brain tissue loss after TBI. To further corroborate the 3D microscopy-based findings, gene expression analyses show that PolySTAT attenuates the expression of inflammation and reactive gliosis biomarkers. These findings support future translational investigation of intravenous PolySTAT as an early post-injury therapy to mitigate neural tissue loss after TBI.

  View details for DOI 10.1126/sciadv.adw7425

  View details for PubMedID 40680138

• Imaging 3D cell cultures with optical microscopy NATURE METHODS Hsieh, H., Han, Q., Brenes, D., Bishop, K. W., Wang, R., Wang, Y., Poudel, C., Glaser, A. K., Freedman, B. S., Vaughan, J. C., Allbritton, N. L., Liu, J. T. C. 2025; 22 (6): 1167-1190

  Abstract

  Three-dimensional (3D) cell cultures have gained popularity in recent years due to their ability to represent complex tissues or organs more faithfully than conventional two-dimensional (2D) cell culture. This article reviews the application of both 2D and 3D microscopy approaches for monitoring and studying 3D cell cultures. We first summarize the most popular optical microscopy methods that have been used with 3D cell cultures. We then discuss the general advantages and disadvantages of various microscopy techniques for several broad categories of investigation involving 3D cell cultures. Finally, we provide perspectives on key areas of technical need in which there are clear opportunities for innovation. Our goal is to guide microscope engineers and biomedical end users toward optimal imaging methods for specific investigational scenarios and to identify use cases in which additional innovations in high-resolution imaging could be helpful.

  View details for DOI 10.1038/s41592-025-02647-w

  View details for Web of Science ID 001468983500001

  View details for PubMedID 40247123

  View details for PubMedCentralID 4341966

• AI-driven Multi-cohort Analysis of 3D Prostate Gland-Skeleton Morphology for Prognosis of Biochemical Recurrence Salguero, J., Medina, S., Serafin, R., Corredor, G., Manandhar, S., Mutha, P., Dhamdhere, R., Wang, R., Chow, S., Romero, E., Bishop, K., Daniel, R., Tokuyama, N., Pathak, T., Mirtti, T., True, L., Lal, P., Liu, J., Madabhushi, A. ELSEVIER SCIENCE INC. 2025

  View details for DOI 10.1016/j.labinv.2024.103122

  View details for Web of Science ID 001469676900032

• Automated, Scalable, and Comprehensive Three-Dimensional Analysis of Glomeruli and Whole Nephrons in Kidney Tissues Poudel, C., Brenes, D., Sandoval, R. M., Martinez-Irizarry, M. M., Dunn, K., Dagher, P. C., Liu, J. T. C., Vaughan, J. C. AMER SOC NEPHROLOGY. 2024

  View details for DOI 10.1681/ASN.2024za0n2xe3

  View details for Web of Science ID 001405917806306

• LiverMap pipeline for 3D imaging of human liver reveals volumetric spatial dysregulation of cirrhotic vasculobiliary architecture. bioRxiv : the preprint server for biology Fabyan, W. B., Fortin, C. L., Kenerson, H. L., Simmonds, S. P., Liu, J. T., Yeh, M. M., Carr, R. M., Yeung, R. S., Stevens, K. R. 2024

  Abstract

  The liver contains an intricate microstructure that is critical for liver function. Architectural disruption of this spatial structure is pathologic. Unfortunately, 2D histopathology - the gold standard for pathological understanding of many liver diseases - can misrepresent or leave gaps in our understanding of complex 3D structural features. Here, we utilized immunostaining, tissue clearing, microscopy, and computational software to create 3D multilobular reconstructions of both non-fibrotic and cirrhotic human liver tissue. We found that spatial architecture in human cirrhotic liver samples with varying etiologies had sinusoid zonation dysregulation, reduction in glutamine synthetase-expressing pericentral hepatocytes, regression of central vein networks, disruption of hepatic arterial networks, and fragmentation of biliary networks, which together suggest a pro-portalization/decentralization phenotype in cirrhotic tissue. Further implementation of 3D pathological analyses may provide a deeper understanding of cirrhotic pathobiology and inspire novel treatments for liver disease.

  View details for DOI 10.1101/2024.09.14.613049

  View details for PubMedID 39345589

  View details for PubMedCentralID PMC11430080

• Axially swept open-top light-sheet microscopy for densely labeled clinical specimens OPTICS LETTERS Bishop, K. W., Barner, L., Baraznenok, E., Lan, L., Poudel, C., Brenes, D., Serafin, R. B., True, L. D., Vaughan, J. C., Glaser, A. K., Liu, J. T. C. 2024; 49 (13): 3794-3797

  Abstract

  Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

  View details for DOI 10.1364/OL.521591

  View details for Web of Science ID 001294036200012

  View details for PubMedID 38950270

• Analysis of 3D pathology samples using weakly supervised AI CELL Song, A. H., Williams, M., Williamson, D. F. K., Chow, S. S. L., Jaume, G., Gao, G., Zhang, A., Chen, B., Baras, A. S., Serafin, R., Colling, R., Downes, M. R., Farre, X., Humphrey, P., Verrill, C., True, L. D., Parwani, A. V., Liu, J. T. C., Mahmood, F. 2024; 187 (10): 2502-2520.e17

  Abstract

  Human tissue, which is inherently three-dimensional (3D), is traditionally examined through standard-of-care histopathology as limited two-dimensional (2D) cross-sections that can insufficiently represent the tissue due to sampling bias. To holistically characterize histomorphology, 3D imaging modalities have been developed, but clinical translation is hampered by complex manual evaluation and lack of computational platforms to distill clinical insights from large, high-resolution datasets. We present TriPath, a deep-learning platform for processing tissue volumes and efficiently predicting clinical outcomes based on 3D morphological features. Recurrence risk-stratification models were trained on prostate cancer specimens imaged with open-top light-sheet microscopy or microcomputed tomography. By comprehensively capturing 3D morphologies, 3D volume-based prognostication achieves superior performance to traditional 2D slice-based approaches, including clinical/histopathological baselines from six certified genitourinary pathologists. Incorporating greater tissue volume improves prognostic performance and mitigates risk prediction variability from sampling bias, further emphasizing the value of capturing larger extents of heterogeneous morphology.

  View details for DOI 10.1016/j.cell.2024.03.035

  View details for Web of Science ID 001240596700001

  View details for PubMedID 38729110

  View details for PubMedCentralID PMC11168832

• Direct three-dimensional segmentation of prostate glands with nnU-Net JOURNAL OF BIOMEDICAL OPTICS Wang, R., Chow, S. S. L., Serafin, R. B., Xie, W., Han, Q., Baraznenok, E., Lan, L., Bishop, K. W., Liu, J. T. C. 2024; 29 (3): 036001

  Abstract

  In recent years, we and others have developed non-destructive methods to obtain three-dimensional (3D) pathology datasets of clinical biopsies and surgical specimens. For prostate cancer risk stratification (prognostication), standard-of-care Gleason grading is based on examining the morphology of prostate glands in thin 2D sections. This motivates us to perform 3D segmentation of prostate glands in our 3D pathology datasets for the purposes of computational analysis of 3D glandular features that could offer improved prognostic performance.To facilitate prostate cancer risk assessment, we developed a computationally efficient and accurate deep learning model for 3D gland segmentation based on open-top light-sheet microscopy datasets of human prostate biopsies stained with a fluorescent analog of hematoxylin and eosin (H&E).For 3D gland segmentation based on our H&E-analog 3D pathology datasets, we previously developed a hybrid deep learning and computer vision-based pipeline, called image translation-assisted segmentation in 3D (ITAS3D), which required a complex two-stage procedure and tedious manual optimization of parameters. To simplify this procedure, we use the 3D gland-segmentation masks previously generated by ITAS3D as training datasets for a direct end-to-end deep learning-based segmentation model, nnU-Net. The inputs to this model are 3D pathology datasets of prostate biopsies rapidly stained with an inexpensive fluorescent analog of H&E and the outputs are 3D semantic segmentation masks of the gland epithelium, gland lumen, and surrounding stromal compartments within the tissue.nnU-Net demonstrates remarkable accuracy in 3D gland segmentations even with limited training data. Moreover, compared with the previous ITAS3D pipeline, nnU-Net operation is simpler and faster, and it can maintain good accuracy even with lower-resolution inputs.Our trained DL-based 3D segmentation model will facilitate future studies to demonstrate the value of computational 3D pathology for guiding critical treatment decisions for patients with prostate cancer.

  View details for DOI 10.1117/1.JBO.29.3.036001

  View details for Web of Science ID 001281200500004

  View details for PubMedID 38434772

  View details for PubMedCentralID PMC10905031

• An end-to-end workflow for nondestructive 3D pathology NATURE PROTOCOLS Bishop, K. W., Erion Barner, L. A., Han, Q., Baraznenok, E., Lan, L., Poudel, C., Gao, G., Serafin, R. B., Chow, S. S. L., Glaser, A. K., Janowczyk, A., Brenes, D., Huang, H., Miyasato, D., True, L. D., Kang, S., Vaughan, J. C., Liu, J. T. C. 2024; 19 (4): 1122-1148

  Abstract

  Recent advances in 3D pathology offer the ability to image orders of magnitude more tissue than conventional pathology methods while also providing a volumetric context that is not achievable with 2D tissue sections, and all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis, however, is not trivial and requires careful attention to a series of details during tissue preparation, imaging and initial data processing, as well as iterative optimization of the entire process. Here, we provide an end-to-end procedure covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. Although 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol focuses on the use of a fluorescent analog of hematoxylin and eosin, which remains the most common stain used for gold-standard pathological reports. We present our guidelines for a broad range of end users (e.g., biologists, clinical researchers and engineers) in a simple format. The end-to-end workflow requires 3-6 d to complete, bearing in mind that data analysis may take longer.

  View details for DOI 10.1038/s41596-023-00934-4

  View details for Web of Science ID 001147322600001

  View details for PubMedID 38263522

  View details for PubMedCentralID 8118147

• Triage of 3D pathology data via 2.5D multiple-instance learning to guide pathologist assessments Gao, G., Song, A. H., Wang, F., Brenes, D., Wang, R., Chow, S. S. L., Bishop, K. W., True, L. D., Mahmood, F., Liu, J. T. C., IEEE IEEE COMPUTER SOC. 2024: 6955-6965

  View details for DOI 10.1109/CVPRW63382.2024.00689

  View details for Web of Science ID 001327781707014

• Artificial Intelligence-Triaged 3-Dimensional Pathology to Improve Detection of Esophageal Neoplasia While Reducing Pathologist Workloads. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Erion Barner, L. A., Gao, G., Reddi, D. M., Lan, L., Burke, W., Mahmood, F., Grady, W. M., Liu, J. T. 2023; 36 (12): 100322

  Abstract

  Early detection of esophageal neoplasia via evaluation of endoscopic surveillance biopsies is the key to maximizing survival for patients with Barrett's esophagus, but it is hampered by the sampling limitations of conventional slide-based histopathology. Comprehensive evaluation of whole biopsies with 3-dimensional (3D) pathology may improve early detection of malignancies, but large 3D pathology data sets are tedious for pathologists to analyze. Here, we present a deep learning-based method to automatically identify the most critical 2-dimensional (2D) image sections within 3D pathology data sets for pathologists to review. Our method first generates a 3D heatmap of neoplastic risk for each biopsy, then classifies all 2D image sections within the 3D data set in order of neoplastic risk. In a clinical validation study, we diagnose esophageal biopsies with artificial intelligence-triaged 3D pathology (3 images per biopsy) vs standard slide-based histopathology (16 images per biopsy) and show that our method improves detection sensitivity while reducing pathologist workloads.

  View details for DOI 10.1016/j.modpat.2023.100322

  View details for PubMedID 37657711

• Visual Assessment of 2-Dimensional Levels Within 3-Dimensional Pathology Data Sets of Prostate Needle Biopsies Reveals Substantial Spatial Heterogeneity LABORATORY INVESTIGATION Koyuncu, C., Janowczyk, A., Farre, X., Pathak, T., Mirtti, T., Fernandez, P. L., Pons, L., Reder, N. P., Sera, R., Chow, S. S. L., Viswanathan, V. S., Glaser, A. K., True, L. D., Liu, J. T. C., Madabhushi, A. 2023; 103 (12): 100265

  Abstract

  Prostate cancer prognostication largely relies on visual assessment of a few thinly sectioned biopsy specimens under a microscope to assign a Gleason grade group (GG). Unfortunately, the assigned GG is not always associated with a patient's outcome in part because of the limited sampling of spatially heterogeneous tumors achieved by 2-dimensional histopathology. In this study, open-top light-sheet microscopy was used to obtain 3-dimensional pathology data sets that were assessed by 4 human readers. Intrabiopsy variability was assessed by asking readers to perform Gleason grading of 5 different levels per biopsy for a total of 20 core needle biopsies (ie, 100 total images). Intrabiopsy variability (Cohen κ) was calculated as the worst pairwise agreement in GG between individual levels within each biopsy and found to be 0.34, 0.34, 0.38, and 0.43 for the 4 pathologists. These preliminary results reveal that even within a 1-mm-diameter needle core, GG based on 2-dimensional images can vary dramatically depending on the location within a biopsy being analyzed. We believe that morphologic assessment of whole biopsies in 3 dimension has the potential to enable more reliable and consistent tumor grading.

  View details for DOI 10.1016/j.labinv.2023.100265

  View details for Web of Science ID 001111301900001

  View details for PubMedID 37858679

  View details for PubMedCentralID PMC10926776

• Engineering the future of 3D pathology JOURNAL OF PATHOLOGY CLINICAL RESEARCH Liu, J. T. C., Chow, S. S. L., Colling, R., Downes, M. R., Farre, X., Humphrey, P., Janowczyk, A., Mirtti, T., Verrill, C., Zlobec, I., True, L. D. 2024; 10 (1): e347

  Abstract

  In recent years, technological advances in tissue preparation, high-throughput volumetric microscopy, and computational infrastructure have enabled rapid developments in nondestructive 3D pathology, in which high-resolution histologic datasets are obtained from thick tissue specimens, such as whole biopsies, without the need for physical sectioning onto glass slides. While 3D pathology generates massive datasets that are attractive for automated computational analysis, there is also a desire to use 3D pathology to improve the visual assessment of tissue histology. In this perspective, we discuss and provide examples of potential advantages of 3D pathology for the visual assessment of clinical specimens and the challenges of dealing with large 3D datasets (of individual or multiple specimens) that pathologists have not been trained to interpret. We discuss the need for artificial intelligence triaging algorithms and explainable analysis methods to assist pathologists or other domain experts in the interpretation of these novel, often complex, large datasets.

  View details for DOI 10.1002/cjp2.347

  View details for Web of Science ID 001095792800001

  View details for PubMedID 37919231

  View details for PubMedCentralID PMC10807588

• Tracking the 3D Architecture of Hundreds of Nephrons and Peritubular Capillaries in Health and Disease Using Light Sheet Microscopy and Deep Learning Poudel, C., Sandoval, R. M., Wong, M. K., Liu, J. T., Vaughan, J. C., Vaughan Lab AMER SOC NEPHROLOGY. 2023: 415

  View details for Web of Science ID 001424476603152

• Miniature line-scanned dual-axis confocal microscope for versatile clinical use BIOMEDICAL OPTICS EXPRESS Bishop, K., Hu, B., Yawhare, R., Yang, Z., Liang, D., Gao, G., Baraznenok, E., Han, Q., Lan, L., Chow, S., Sanai, N., Liu, J. 2023; 14 (11): 6048-6059

  Abstract

  A miniature optical-sectioning fluorescence microscope with high sensitivity and resolution would enable non-invasive and real-time tissue inspection, with potential use cases including early disease detection and intraoperative guidance. Previously, we developed a miniature MEMS-based dual-axis confocal (DAC) microscope that enabled video-rate optically sectioned in vivo microscopy of human tissues. However, the device's clinical utility was limited due to a small field of view, a non-adjustable working distance, and a lack of a sterilization strategy. In our latest design, we have made improvements to achieve a 2x increase in the field of view (600 × 300 µm) and an adjustable working distance range of 150 µm over a wide range of excitation/emission wavelengths (488-750 nm), all while maintaining a high frame rate of 15 frames per second (fps). Furthermore, the device is designed to image through a disposable sterile plastic drape for convenient clinical use. We rigorously characterize the performance of the device and show example images of ex vivo tissues to demonstrate the optical performance of our new design, including fixed mouse skin and human prostate, as well as fresh mouse kidney, mouse intestine, and human head and neck surgical specimens with corresponding H&E histology. These improvements will facilitate clinical testing and translation.

  View details for DOI 10.1364/BOE.503478

  View details for Web of Science ID 001104188200002

  View details for PubMedID 38021137

  View details for PubMedCentralID PMC10659777

• Nondestructive 3D Pathology Image Atlas of Barrett Esophagus With Open-Top Light-Sheet Microscopy ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Reddi, D. M., Barner, L. A., Burke, W., Gao, G., Grady, W. M., Liu, J. T. C. 2023; 147 (10): 1164-1171

  Abstract

  Anatomic pathologists render diagnosis on tissue samples sectioned onto glass slides and viewed under a bright-field microscope. This approach is destructive to the sample, which can limit its use for ancillary assays that can inform patient management. Furthermore, the subjective interpretation of a relatively small number of 2D tissue sections per sample contributes to low interobserver agreement among pathologists for the assessment (diagnosis and grading) of various lesions.To evaluate 3D pathology data sets of thick formalin-fixed Barrett esophagus specimens imaged nondestructively with open-top light-sheet (OTLS) microscopy.Formalin-fixed, paraffin-embedded Barrett esophagus samples (N = 15) were deparaffinized, stained with a fluorescent analog of hematoxylin-eosin, optically cleared, and imaged nondestructively with OTLS microscopy. The OTLS microscopy images were subsequently compared with archived hematoxylin-eosin histology sections from each sample.Barrett esophagus samples, both small endoscopic forceps biopsies and endoscopic mucosal resections, exhibited similar resolvable structures between OTLS microscopy and conventional light microscopy with up to a ×20 objective (×200 overall magnification). The 3D histologic images generated by OTLS microscopy can enable improved discrimination of cribriform and well-formed gland morphologies. In addition, a much larger amount of tissue is visualized with OTLS microscopy, which enables improved assessment of clinical specimens exhibiting high spatial heterogeneity.In esophageal specimens, OTLS microscopy can generate images comparable in quality to conventional light microscopy, with the advantages of providing 3D information for enhanced evaluation of glandular morphologies and enabling much more of the tissue specimen to be visualized nondestructively.

  View details for DOI 10.5858/arpa.2022-0133-OA

  View details for Web of Science ID 001108713600003

  View details for PubMedID 36596255

• An end-to-end workflow for non-destructive 3D pathology. bioRxiv : the preprint server for biology Bishop, K. W., Barner, L. A., Han, Q., Baraznenok, E., Lan, L., Poudel, C., Gao, G., Serafin, R. B., Chow, S. S., Glaser, A. K., Janowczyk, A., Brenes, D., Huang, H., Miyasato, D., True, L. D., Kang, S., Vaughan, J. C., Liu, J. T. 2023

  Abstract

  Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.

  View details for DOI 10.1101/2023.08.03.551845

  View details for PubMedID 37577615

• Weakly Supervised AI for Efficient Analysis of 3D Pathology Samples. ArXiv Song, A. H., Williams, M., Williamson, D. F., Jaume, G., Zhang, A., Chen, B., Serafin, R., Liu, J. T., Baras, A., Parwani, A. V., Mahmood, F. 2023

  Abstract

  Human tissue consists of complex structures that display a diversity of morphologies, forming a tissue microenvironment that is, by nature, three-dimensional (3D). However, the current standard-of-care involves slicing 3D tissue specimens into two-dimensional (2D) sections and selecting a few for microscopic evaluation1,2, with concomitant risks of sampling bias and misdiagnosis3-6. To this end, there have been intense efforts to capture 3D tissue morphology and transition to 3D pathology, with the development of multiple high-resolution 3D imaging modalities7-18. However, these tools have had little translation to clinical practice as manual evaluation of such large data by pathologists is impractical and there is a lack of computational platforms that can efficiently process the 3D images and provide patient-level clinical insights. Here we present Modality-Agnostic Multiple instance learning for volumetric Block Analysis (MAMBA), a deep-learning-based platform for processing 3D tissue images from diverse imaging modalities and predicting patient outcomes. Archived prostate cancer specimens were imaged with open-top light-sheet microscopy12-14 or microcomputed tomography15,16 and the resulting 3D datasets were used to train risk-stratification networks based on 5-year biochemical recurrence outcomes via MAMBA. With the 3D block-based approach, MAMBA achieves an area under the receiver operating characteristic curve (AUC) of 0.86 and 0.74, superior to 2D traditional single-slice-based prognostication (AUC of 0.79 and 0.57), suggesting superior prognostication with 3D morphological features. Further analyses reveal that the incorporation of greater tissue volume improves prognostic performance and mitigates risk prediction variability from sampling bias, suggesting that there is value in capturing larger extents of spatially heterogeneous 3D morphology. With the rapid growth and adoption of 3D spatial biology and pathology techniques by researchers and clinicians, MAMBA provides a general and efficient framework for 3D weakly supervised learning for clinical decision support and can help to reveal novel 3D morphological biomarkers for prognosis and therapeutic response.

  View details for PubMedID 37547660

• Comprehensive Surface Histology of Fresh Resection Margins With Rapid Open-Top Light-Sheet (OTLS) Microscopy IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Gao, G., Miyasato, D., Barner, L. A., Serafin, R., Bishop, K. W., Xie, W., Glaser, A. K., Rosenthal, E. L., True, L. D., Liu, J. T. C. 2023; 70 (7): 2160-2171

  Abstract

  For tumor resections, margin status typically correlates with patient survival but positive margin rates are generally high (up to 45% for head and neck cancer). Frozen section analysis (FSA) is often used to intraoperatively assess the margins of excised tissue, but suffers from severe under-sampling of the actual margin surface, inferior image quality, slow turnaround, and tissue destructiveness.Here, we have developed an imaging workflow to generate en face histologic images of freshly excised surgical margin surfaces based on open-top light-sheet (OTLS) microscopy. Key innovations include (1) the ability to generate false-colored H&E-mimicking images of tissue surfaces stained for < 1 min with a single fluorophore, (2) rapid OTLS surface imaging at a rate of 15 min/cm2 followed by real-time post-processing of datasets within RAM at a rate of 5 min/cm2, and (3) rapid digital surface extraction to account for topological irregularities at the tissue surface.In addition to the performance metrics listed above, we show that the image quality generated by our rapid surface-histology method approaches that of gold-standard archival histology.OTLS microscopy has the feasibility to provide intraoperative guidance of surgical oncology procedures.The reported methods can potentially improve tumor-resection procedures, thereby improving patient outcomes and quality of life.

  View details for DOI 10.1109/TBME.2023.3237267

  View details for Web of Science ID 001016857400017

  View details for PubMedID 37021859

  View details for PubMedCentralID PMC10324671

• 3D open-top light-sheet microscopy and 3D microdissection of neuoadjuvant-treated primary prostate cancer reveals latent subclonal mutations. Reder, N., Pritchard, C. C., Konnick, E. Q., Huang, H., Lerma, A., Glaser, A. K., True, L. D., Liu, J. T. C., Schweizer, M. LIPPINCOTT WILLIAMS & WILKINS. 2023

  View details for Web of Science ID 001053772002556

• Nondestructive 3D pathology with analysis of nuclear features for prostate cancer risk assessment JOURNAL OF PATHOLOGY Serafin, R., Koyuncu, C., Xie, W., Huang, H., Glaser, A. K., Reder, N. P., Janowczyk, A., True, L. D., Madabhushi, A., Liu, J. T. C. 2023; 260 (4): 390-401

  Abstract

  Prostate cancer treatment decisions rely heavily on subjective visual interpretation [assigning Gleason patterns or International Society of Urological Pathology (ISUP) grade groups] of limited numbers of two-dimensional (2D) histology sections. Under this paradigm, interobserver variance is high, with ISUP grades not correlating well with outcome for individual patients, and this contributes to the over- and undertreatment of patients. Recent studies have demonstrated improved prognostication of prostate cancer outcomes based on computational analyses of glands and nuclei within 2D whole slide images. Our group has also shown that the computational analysis of three-dimensional (3D) glandular features, extracted from 3D pathology datasets of whole intact biopsies, can allow for improved recurrence prediction compared to corresponding 2D features. Here we seek to expand on these prior studies by exploring the prognostic value of 3D shape-based nuclear features in prostate cancer (e.g. nuclear size, sphericity). 3D pathology datasets were generated using open-top light-sheet (OTLS) microscopy of 102 cancer-containing biopsies extracted ex vivo from the prostatectomy specimens of 46 patients. A deep learning-based workflow was developed for 3D nuclear segmentation within the glandular epithelium versus stromal regions of the biopsies. 3D shape-based nuclear features were extracted, and a nested cross-validation scheme was used to train a supervised machine classifier based on 5-year biochemical recurrence (BCR) outcomes. Nuclear features of the glandular epithelium were found to be more prognostic than stromal cell nuclear features (area under the ROC curve [AUC] = 0.72 versus 0.63). 3D shape-based nuclear features of the glandular epithelium were also more strongly associated with the risk of BCR than analogous 2D features (AUC = 0.72 versus 0.62). The results of this preliminary investigation suggest that 3D shape-based nuclear features are associated with prostate cancer aggressiveness and could be of value for the development of decision-support tools. © 2023 The Pathological Society of Great Britain and Ireland.

  View details for DOI 10.1002/path.6090

  View details for Web of Science ID 000994842700001

  View details for PubMedID 37232213

  View details for PubMedCentralID PMC10524574

• Innovations in Remote Teaching of Engineering Design Teams. Annual Conference & Exposition : final program and proceedings. American Society for Engineering Education Kang, S., Blakeney, E. A., Yasuhara, K., Kearney, K. E., Payne, S., Seibel, E., Liu, J. T., Reinhall, P., Posner, J. 2023; 2023

  Abstract

  The University of Washington's Engineering Innovation in Health program is a yearlong engineering design course sequence where senior undergraduate and graduate engineering students across different disciplines work in teams with health professionals to address their unmet needs. With the onset of the COVID-19 pandemic, these team- and project-based courses shifted from an in-person to remote course environment. Here, we share innovative teaching strategies for a team-based, remote course environment. We show how this shift affected productivity by comparing survey results from before (in person) and during (remote) the pandemic. Preliminary results show that overall project outcomes and productivity were as high or, in some cases, higher during the pandemic than prior to the pandemic. These findings suggest that the innovative remote teaching strategies implemented by the teaching team provided effective options in the absence of certain hands-on experiences that are considered critical to engineering capstone design courses. A discussion on these teaching strategies in the context beyond the pandemic are considered in the discussion.

  View details for PubMedID 38187933

• Introduction to the Biophotonics Congress 2022 feature issue BIOMEDICAL OPTICS EXPRESS Liu, J. T. C., Bale, G., Choe, R., Elson, D. S., Oldenburg, A., Ian, L., Tkaczyk, E. R. 2023; 14 (1): 385-386

  Abstract

  A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optica Biophotonics Congress: Biomedical Optics held on April 24-27, 2022 in Fort Lauderdale, Florida, USA.

  View details for DOI 10.1364/BOE.483553

  View details for Web of Science ID 000914710500004

  View details for PubMedID 36698666

  View details for PubMedCentralID PMC9842003

• Nondestructive 3D Pathology with Light-Sheet Fluorescence Microscopy for Translational Research and Clinical Assays ANNUAL REVIEW OF ANALYTICAL CHEMISTRY Liu, J. T. C., Glaser, A. K., Poudel, C., Vaughan, J. C. 2023; 16: 231-252

  Abstract

  In recent years, there has been a revived appreciation for the importance of spatial context and morphological phenotypes for both understanding disease progression and guiding treatment decisions. Compared with conventional 2D histopathology, which is the current gold standard of medical diagnostics, nondestructive 3D pathology offers researchers and clinicians the ability to visualize orders of magnitude more tissue within their natural volumetric context. This has been enabled by rapid advances in tissue-preparation methods, high-throughput 3D microscopy instrumentation, and computational tools for processing these massive feature-rich data sets. Here, we provide a brief overview of many of these technical advances along with remaining challenges to be overcome. We also speculate on the future of 3D pathology as applied in translational investigations, preclinical drug development, and clinical decision-support assays.

  View details for DOI 10.1146/annurev-anchem-091222-092734

  View details for Web of Science ID 001004789300011

  View details for PubMedID 36854208

• A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues NATURE METHODS Glaser, A. K., Bishop, K. W., Barner, L. A., Susaki, E. A., Kubota, S. I., Gao, G., Serafin, R. B., Balaram, P., Turschak, E., Nicovich, P. R., Lai, H., Lucas, L. A. G., Yi, Y., Nichols, E. K., Huang, H., Reder, N. P., Wilson, J. J., Sivakumar, R., Shamskhou, E., Stoltzfus, C. R., Wei, X., Hempton, A. K., Pende, M., Murawala, P., Dodt, H., Imaizumi, T., Shendure, J., Beliveau, B. J., Gerner, M. Y., Xin, L., Zhao, H., True, L. D., Reid, R., Chandrashekar, J., Ueda, H. R., Svoboda, K., Liu, J. T. C. 2022; 19 (5): 613-+

  Abstract

  Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

  View details for DOI 10.1038/s41592-022-01468-5

  View details for Web of Science ID 000793688400007

  View details for PubMedID 35545715

  View details for PubMedCentralID PMC9214839

• <i>In vivo</i> microscopy as an adjunctive tool to guide detection, diagnosis, and treatment JOURNAL OF BIOMEDICAL OPTICS Bishop, K. W., Maitland, K. C., Rajadhyaksha, M., Liu, J. T. C. 2022; 27 (4)

  Abstract

  There have been numerous academic and commercial efforts to develop high-resolution in vivo microscopes for a variety of clinical use cases, including early disease detection and surgical guidance. While many high-profile studies, commercialized products, and publications have resulted from these efforts, mainstream clinical adoption has been relatively slow other than for a few clinical applications (e.g., dermatology).Here, our goals are threefold: (1) to introduce and motivate the need for in vivo microscopy (IVM) as an adjunctive tool for clinical detection, diagnosis, and treatment, (2) to discuss the key translational challenges facing the field, and (3) to propose best practices and recommendations to facilitate clinical adoption.We will provide concrete examples from various clinical domains, such as dermatology, oral/gastrointestinal oncology, and neurosurgery, to reinforce our observations and recommendations.While the incremental improvement and optimization of IVM technologies should and will continue to occur, future translational efforts would benefit from the following: (1) integrating clinical and industry partners upfront to define and maintain a compelling value proposition, (2) identifying multimodal/multiscale imaging workflows, which are necessary for success in most clinical scenarios, and (3) developing effective artificial intelligence tools for clinical decision support, tempered by a realization that complete adoption of such tools will be slow.The convergence of imaging modalities, academic-industry-clinician partnerships, and new computational capabilities has the potential to catalyze rapid progress and adoption of IVM in the next few decades.

  View details for DOI 10.1117/1.JBO.27.4.040601

  View details for Web of Science ID 000789615800001

  View details for PubMedID 35478042

  View details for PubMedCentralID PMC9043840

• 3D Light-Sheet Microscopy and Microdissection of Primary Prostate Cancer Reveals Important Latent Sub-clonal Mutations Lerma, L., Pritchard, C., Konnick, E., Haffner, M., Schweizer, M., Huang, H., Glaser, A., True, L., Liu, J., Reder, N. SPRINGERNATURE. 2022: 620

  View details for Web of Science ID 000770361801206

• Multiresolution nondestructive 3D pathology of whole lymph nodes for breast cancer staging JOURNAL OF BIOMEDICAL OPTICS Barner, L. A., Glaser, A. K., Mao, C., Susaki, E. A., Vaughan, J. C., Dintzis, S. M., Liu, J. T. C. 2022; 27 (3)

  Abstract

  For breast cancer patients, the extent of regional lymph node (LN) metastasis influences the decision to remove all axillary LNs. Metastases are currently identified and classified with visual analysis of a few thin tissue sections with conventional histology that may underrepresent the extent of metastases.We sought to enable nondestructive three-dimensional (3D) pathology of human axillary LNs and to develop a practical workflow for LN staging with our method. We also sought to evaluate whether 3D pathology improves staging accuracy in comparison to two-dimensional (2D) histology.We developed a method to fluorescently stain and optically clear LN specimens for comprehensive imaging with multiresolution open-top light-sheet microscopy. We present an efficient imaging and data-processing workflow for rapid evaluation of H&E-like datasets in 3D, with low-resolution screening to identify potential metastases followed by high-resolution localized imaging to confirm malignancy.We simulate LN staging with 3D and 2D pathology datasets from 10 metastatic nodes, showing that 2D pathology consistently underestimates metastasis size, including instances in which 3D pathology would lead to upstaging of the metastasis with important implications on clinical treatment.Our 3D pathology method may improve clinical management for breast cancer patients by improving staging accuracy of LN metastases.

  View details for DOI 10.1117/1.JBO.27.3.036501

  View details for Web of Science ID 000776555200007

  View details for PubMedID 35315258

  View details for PubMedCentralID PMC8936940

• Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy NATURE PROTOCOLS Lee, M., Mao, C., Glaser, A. K., Woodworth, M. A., Halpern, A. R., Ali, A., Liu, J. T. C., Vaughan, J. C. 2022; 17 (3): 819-+

  Abstract

  Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid-Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.

  View details for DOI 10.1038/s41596-021-00667-2

  View details for Web of Science ID 000749965800004

  View details for PubMedID 35110740

  View details for PubMedCentralID PMC11404980

• Prostate Cancer Risk Stratification via Nondestructive 3D Pathology with Deep Learning-Assisted Gland Analysis CANCER RESEARCH Xie, W., Reder, N. P., Koyuncu, C., Leo, P., Hawley, S., Huang, H., Mao, C., Postupna, N., Kang, S., Serafin, R., Gao, G., Han, Q., Bishop, K. W., Barner, L. A., Fu, P., Wright, J. L., Keene, C., Vaughan, J. C., Janowczyk, A., Glaser, A. K., Madabhushi, A., True, L. D., Liu, J. T. C. 2022; 82 (2): 334-345

  Abstract

  Prostate cancer treatment planning is largely dependent upon examination of core-needle biopsies. The microscopic architecture of the prostate glands forms the basis for prognostic grading by pathologists. Interpretation of these convoluted three-dimensional (3D) glandular structures via visual inspection of a limited number of two-dimensional (2D) histology sections is often unreliable, which contributes to the under- and overtreatment of patients. To improve risk assessment and treatment decisions, we have developed a workflow for nondestructive 3D pathology and computational analysis of whole prostate biopsies labeled with a rapid and inexpensive fluorescent analogue of standard hematoxylin and eosin (H&E) staining. This analysis is based on interpretable glandular features and is facilitated by the development of image translation-assisted segmentation in 3D (ITAS3D). ITAS3D is a generalizable deep learning-based strategy that enables tissue microstructures to be volumetrically segmented in an annotation-free and objective (biomarker-based) manner without requiring immunolabeling. As a preliminary demonstration of the translational value of a computational 3D versus a computational 2D pathology approach, we imaged 300 ex vivo biopsies extracted from 50 archived radical prostatectomy specimens, of which, 118 biopsies contained cancer. The 3D glandular features in cancer biopsies were superior to corresponding 2D features for risk stratification of patients with low- to intermediate-risk prostate cancer based on their clinical biochemical recurrence outcomes. The results of this study support the use of computational 3D pathology for guiding the clinical management of prostate cancer. SIGNIFICANCE: An end-to-end pipeline for deep learning-assisted computational 3D histology analysis of whole prostate biopsies shows that nondestructive 3D pathology has the potential to enable superior prognostic stratification of patients with prostate cancer.

  View details for DOI 10.1158/0008-5472.CAN-21-2843

  View details for Web of Science ID 000745022100001

  View details for PubMedID 34853071

  View details for PubMedCentralID PMC8803395

• Implementation and evaluation of team science training for interdisciplinary teams in an engineering design program JOURNAL OF CLINICAL AND TRANSLATIONAL SCIENCE Blakeney, E., Kang, S., Henrikson, K., Liu, J. T. C., Seibel, E. J., Sprecher, J., Summerside, N., Vogel, M. T., Zierler, B. K., Posner, J. D. 2021; 5 (1): e127

  Abstract

  Interdisciplinary academic teams perform better when competent in teamwork; however, there is a lack of best practices of how to introduce and facilitate the development of effective learning and functioning within these teams in academic environments.To close this gap, we tailored, implemented, and evaluated team science training in the year-long Engineering Innovation in Health (EIH) program at the University of Washington (UW), a project-based course in which engineering students across several disciplines partner with health professionals to develop technical solutions to clinical and translational health challenges. EIH faculty from the UW College of Engineering and the Institute of Translational Health Sciences' (ITHS) Team Science Core codeveloped and delivered team science training sessions and evaluated their impact with biannual surveys. A student cohort was surveyed prior to the implementation of the team science trainings, which served as a baseline.Survey responses were compared within and between both cohorts (approximately 55 students each Fall Quarter and 30 students each Spring Quarter). Statistically significant improvements in measures of self-efficacy and interpersonal team climate (i.e., psychological safety) were observed within and between teams.Tailored team science training provided to student-professional teams resulted in measurable improvements in self-efficacy and interpersonal climate both of which are crucial for teamwork and intellectual risk taking. Future research is needed to determine long-term impacts of course participation on individual and team outcomes (e.g., patents, start-ups). Additionally, adaptability of this model to clinical and translational research teams in alternate formats and settings should be tested.

  View details for DOI 10.1017/cts.2021.788

  View details for Web of Science ID 000688496100002

  View details for PubMedID 34367672

  View details for PubMedCentralID PMC8327544

• Intraoperative Fluorescence-Guided Surgery in Head and Neck Squamous Cell Carcinoma. The Laryngoscope Lee, Y. J., Krishnan, G., Nishio, N., van den Berg, N. S., Lu, G., Martin, B. A., van Keulen, S., Colevas, A. D., Kapoor, S., Liu, J. T., Rosenthal, E. L. 2021; 131 (3): 529-534

  Abstract

  The rate of positive margins in head and neck cancers has remained stagnant over the past three decades and is consistently associated with poor overall survival. This suggests that significant improvements must be made intraoperatively to ensure negative margins. We discuss the important role of fluorescence imaging to guide surgical oncology in head and neck cancer. This review includes a general overview of the principles of fluorescence, available fluorophores used for fluorescence imaging, and specific clinical applications of fluorescence-guided surgery, as well as challenges and future directions in head and neck surgical oncology. Laryngoscope, 131:529-534, 2021.

  View details for DOI 10.1002/lary.28822

  View details for PubMedID 33593036

• Harnessing non-destructive 3D pathology NATURE BIOMEDICAL ENGINEERING Liu, J. T. C., Glaser, A. K., Bera, K., True, L. D., Reder, N. P., Eliceiri, K. W., Madabhushi, A. 2021; 5 (3): 203-218

  Abstract

  High-throughput methods for slide-free three-dimensional (3D) pathological analyses of whole biopsies and surgical specimens offer the promise of modernizing traditional histology workflows and delivering improvements in diagnostic performance. Advanced optical methods now enable the interrogation of orders of magnitude more tissue than previously possible, where volumetric imaging allows for enhanced quantitative analyses of cell distributions and tissue structures that are prognostic and predictive. Non-destructive imaging processes can simplify laboratory workflows, potentially reducing costs, and can ensure that samples are available for subsequent molecular assays. However, the large size of the feature-rich datasets that they generate poses challenges for data management and computer-aided analysis. In this Perspective, we provide an overview of the imaging technologies that enable 3D pathology, and the computational tools-machine learning, in particular-for image processing and interpretation. We also discuss the integration of various other diagnostic modalities with 3D pathology, along with the challenges and opportunities for clinical adoption and regulatory approval.

  View details for DOI 10.1038/s41551-020-00681-x

  View details for Web of Science ID 000618171800001

  View details for PubMedID 33589781

  View details for PubMedCentralID PMC8118147

• Microdissected "cuboids" for microfluidic drug testing of intact tissues LAB ON A CHIP Horowitz, L. F., Rodriguez, A. D., Au-Yeung, A., Bishop, K. W., Barner, L. A., Mishra, G., Raman, A., Delgado, P., Liu, J. T. C., Gujral, T. S., Mehrabi, M., Yang, M., Pierce, R. H., Folch, A. 2021; 21 (1): 122-142

  Abstract

  As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

  View details for DOI 10.1039/d0lc00801j

  View details for Web of Science ID 000607297200006

  View details for PubMedID 33174580

  View details for PubMedCentralID PMC8205430

• Three-dimensional histo-morphometric features from light-sheet microscopy images result in improved discrimination of benign from malignant glands in prostate cancer Koyuncu, C. F., Janowczyk, A., Lu, C., Leo, P., Alilou, M., Glaser, A. K., Reder, N. P., Liu, J. T. C., Madabhushi, A. edited by Tomaszewski, J. E., Ward, A. D. SPIE-INT SOC OPTICAL ENGINEERING. 2021

  View details for DOI 10.1117/12.2549726

  View details for Web of Science ID 000672555600014

• Diagnosing 12 prostate needle cores within an hour of biopsy via open-top light-sheet microscopy JOURNAL OF BIOMEDICAL OPTICS Xie, W., Glaser, A. K., Vakar-Lopez, F., Wright, J. L., Reder, N. P., Liu, J. T. C., True, L. D. 2020; 25 (12)

  Abstract

  Processing and diagnosing a set of 12 prostate biopsies using conventional histology methods typically take at least one day. A rapid and accurate process performed while the patient is still on-site could significantly improve the patient's quality of life.We develop and assess the feasibility of a one-hour-to-diagnosis (1Hr2Dx) method for processing and providing a preliminary diagnosis of a set of 12 prostate biopsies.We developed a fluorescence staining, optical clearing, and 3D open-top light-sheet microscopy workflow to enable 12 prostate needle core biopsies to be processed and diagnosed within an hour of receipt. We analyzed 44 biopsies by the 1Hr2Dx method, which does not consume tissue. The biopsies were then processed for routine, slide-based 2D histology. Three pathologists independently evaluated the 3D 1Hr2Dx and 2D slide-based datasets in a blinded, randomized fashion. Turnaround times were recorded, and the accuracy of our method was compared with gold-standard slide-based histology.The average turnaround time for tissue processing, imaging, and diagnosis was 44.5 min. The sensitivity and specificity of 1Hr2Dx in diagnosing cancer were both >90  %  .The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy.

  View details for DOI 10.1117/1.JBO.25.12.126502

  View details for Web of Science ID 000605144900016

  View details for PubMedID 33325186

  View details for PubMedCentralID PMC7744172

• Multi-resolution open-top light-sheet microscopy to enable efficient 3D pathology workflows BIOMEDICAL OPTICS EXPRESS Barner, L. A., Glaser, A. K., Huang, H., True, L. D., Liu, J. T. C. 2020; 11 (11): 6605-6619

  Abstract

  Open-top light-sheet (OTLS) microscopes have been developed for user-friendly and versatile high-throughput 3D microscopy of thick specimens. As with all imaging modalities, spatial resolution trades off with imaging and analysis times. A hierarchical multi-scale imaging workflow would therefore be of value for many volumetric microscopy applications. We describe a compact multi-resolution OTLS microscope, enabled by a novel solid immersion meniscus lens (SIMlens), which allows users to rapidly transition between air-based objectives for low- and high-resolution 3D imaging. We demonstrate the utility of this system by showcasing an efficient 3D analysis workflow for a diagnostic pathology application.

  View details for DOI 10.1364/BOE.408684

  View details for Web of Science ID 000583376400038

  View details for PubMedID 33282511

  View details for PubMedCentralID PMC7687944

• FalseColor-Python: A rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology PLOS ONE Serafin, R., Xie, W., Glaser, A. K., Liu, J. T. C. 2020; 15 (10): e0233198

  Abstract

  Slide-free digital pathology techniques, including nondestructive 3D microscopy, are gaining interest as alternatives to traditional slide-based histology. In order to facilitate clinical adoption of these fluorescence-based techniques, software methods have been developed to convert grayscale fluorescence images into color images that mimic the appearance of standard absorptive chromogens such as hematoxylin and eosin (H&E). However, these false-coloring algorithms often require manual and iterative adjustment of parameters, with results that can be inconsistent in the presence of intensity nonuniformities within an image and/or between specimens (intra- and inter-specimen variability). Here, we present an open-source (Python-based) rapid intensity-leveling and digital-staining package that is specifically designed to render two-channel fluorescence images (i.e. a fluorescent analog of H&E) to the traditional H&E color space for 2D and 3D microscopy datasets. However, this method can be easily tailored for other false-coloring needs. Our package offers (1) automated and uniform false coloring in spite of uneven staining within a large thick specimen, (2) consistent color-space representations that are robust to variations in staining and imaging conditions between different specimens, and (3) GPU-accelerated data processing to allow these methods to scale to large datasets. We demonstrate this platform by generating H&E-like images from cleared tissues that are fluorescently imaged in 3D with open-top light-sheet (OTLS) microscopy, and quantitatively characterizing the results in comparison to traditional slide-based H&E histology.

  View details for DOI 10.1371/journal.pone.0233198

  View details for Web of Science ID 000590270000005

  View details for PubMedID 33001995

  View details for PubMedCentralID PMC7529223

• Video-Mosaicked Handheld Dual-Axis Confocal Microscopy of Gliomas: An<i>ex vivo</i>Feasibility Study in Humans FRONTIERS IN ONCOLOGY Fujita, Y., Wei, L., Cimino, P. J., Liu, J. T. C., Sanai, N. 2020; 10: 1674

  Abstract

  Intraoperative confocal microscopy can enable high-resolution cross-sectional imaging of intact tissues as a non-invasive real-time alternative to gold-standard histology. However, all current means of intraoperative confocal microscopy are hindered by a limited field of view (FOV), presenting a challenge for evaluating gliomas, which are highly heterogeneous.This study explored the use of image mosaicking with handheld dual-axis confocal (DAC) microscopy of fresh human glioma specimens.In this preliminary technical feasibility study, fresh human glioma specimens from 6 patients were labeled with a fast-acting topical stain (acridine orange) and imaged using a newly developed DAC microscope prototype.In comparison to individual image frames with small fields of view, mosaicked images from a DAC microscope correlate better with gold-standard H&E-stained histology images, including the ability to visualize gradual transitions from areas of dense cellularity to sparse cellularity within the tumor.LS-DAC microscopy provides high-resolution, high-contrast images of glioma tissues that agree with corresponding H&E histology. Compared with individual image frames, mosaicked images provide more accurate representations of the overall cytoarchitecture of heterogeneous glioma tissues. Further investigation is needed to evaluate the ability of high-resolution mosaicked microscopy to improve the extent of glioma resection and patient outcomes.

  View details for DOI 10.3389/fonc.2020.01674

  View details for Web of Science ID 000570390600001

  View details for PubMedID 32974207

  View details for PubMedCentralID PMC7482651

• Performance tradeoffs for single- and dual-objective open-top light-sheet microscope designs: a simulation-based analysis BIOMEDICAL OPTICS EXPRESS Bishop, K. W., Glaser, A. K., Liu, J. T. C. 2020; 11 (8): 4627-4650

  Abstract

  Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design tradeoffs for LSM systems, we present a diffraction-based analysis of single- and dual-objective LSM configurations using simulations of LSM point spread functions. We assume Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8).

  View details for DOI 10.1364/BOE.397052

  View details for Web of Science ID 000577451600041

  View details for PubMedID 32923068

  View details for PubMedCentralID PMC7449713

• Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy SCIENCE ADVANCES Mao, C., Lee, M., Jhan, J., Halpern, A. R., Woodworth, M. A., Glaser, A. K., Chozinski, T. J., Shin, L., Pippin, J. W., Shankland, S. J., Liu, J., Vaughan, J. C. 2020; 6 (22): eaba4542

  Abstract

  Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

  View details for DOI 10.1126/sciadv.aba4542

  View details for Web of Science ID 000537238200043

  View details for PubMedID 32518827

  View details for PubMedCentralID PMC7253160

• Real-time video mosaicking to guide handheld in vivo microscopy JOURNAL OF BIOPHOTONICS Yin, C., Wei, L., Kose, K., Glaser, A. K., Peterson, G., Rajadhyaksha, M., Liu, J. T. C. 2020; 13 (6): e202000048

  Abstract

  Handheld and endoscopic optical-sectioning microscopes are being developed for noninvasive screening and intraoperative consultation. Imaging a large extent of tissue is often desired, but miniature in vivo microscopes tend to suffer from limited fields of view. To extend the imaging field during clinical use, we have developed a real-time video mosaicking method, which allows users to efficiently survey larger areas of tissue. Here, we modified a previous post-processing mosaicking method so that real-time mosaicking is possible at >30 frames/second when using a device that outputs images that are 400 × 400 pixels in size. Unlike other real-time mosaicking methods, our strategy can accommodate image rotations and deformations that often occur during clinical use of a handheld microscope. We perform a feasibility study to demonstrate that the use of real-time mosaicking is necessary to enable efficient sampling of a desired imaging field when using a handheld dual-axis confocal microscope.

  View details for DOI 10.1002/jbio.202000048

  View details for Web of Science ID 000525905500001

  View details for PubMedID 32246558

  View details for PubMedCentralID PMC7969124

• Diagnosis of All 12 Needle Cores within an Hour of a Prostate Biopsy Procedure Xie, W., Reder, N., Vakar-Lopez, F., Liu, J., Glaser, A., True, L. SPRINGERNATURE. 2020: 992-993

  View details for Web of Science ID 000518328902177

• 3D Open-Top Light-Sheet (OTLS) Fluorescence Microscopy of Entire Prostate Core Biopsies Improves Diagnostic Accuracy Malleis, J., Glaser, A., Kang, S., Pang, K., True, L., Liu, J., Reder, N. SPRINGERNATURE. 2020: 929

  View details for Web of Science ID 000518328902106

• Light-Sheet Microscopy for 3D Pathology of a Variety of Human Tissues Glaser, A., Reder, N., True, L., Liu, J. SPRINGERNATURE. 2020: 1697-1698

  View details for Web of Science ID 000518328903495

• Multi-immersion open-top light-sheet microscopy Glaser, A. K., Liu, J. T. C. edited by Brown, T. G., Wilson, T., Waller, L. SPIE-INT SOC OPTICAL ENGINEERING. 2020

  View details for DOI 10.1117/12.2543116

  View details for Web of Science ID 000558351200002

• INTRAOPERATIVE HAND-HELD LINE-SCANNED DUAL-AXIS CONFOCAL MICROSCOPY FOR VISUALIZING LOW-GRADE GLIOMAS Fujita, Y., Wei, L., Liu, J. T. C., Sanai, N. OXFORD UNIV PRESS INC. 2019: 240

  View details for Web of Science ID 000509478706017

• Solid immersion meniscus lens (SIMlens) for open-top light-sheet microscopy OPTICS LETTERS Barner, L. A., Glaser, A. K., True, L. D., Reder, N. P., Liu, J. T. C. 2019; 44 (18): 4451-4454

  Abstract

  Open-top light-sheet (OTLS) microscopy has been developed for rapid volumetric imaging of large pathology specimens. A challenge with OTLS microscopy is the transmission of oblique illumination and detection beams through a horizontal sample plate without introducing aberrations. Previous solutions prevented vertical sample movement, constrained the refractive index of the sample, and/or hindered multi-resolution imaging. Here we describe a solid immersion meniscus lens, a wavefront-matching element that suppresses aberrations when illumination and detection beam transition between air and various high-index immersion media, thereby enabling multi-resolution OTLS microscopy of specimens cleared with diverse protocols.

  View details for DOI 10.1364/OL.44.004451

  View details for Web of Science ID 000485817500005

  View details for PubMedID 31517904

  View details for PubMedCentralID PMC7331451

• Open-Top Light-Sheet Microscopy Image Atlas of Prostate Core Needle Biopsies ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Reder, N. P., Glaser, A. K., McCarty, E. F., Chen, Y., True, L. D., Liu, J. T. C. 2019; 143 (9): 1069-1075

  Abstract

  Ex vivo microscopy encompasses a range of techniques to examine fresh or fixed tissue with microscopic resolution, eliminating the need to embed the tissue in paraffin or produce a glass slide. One such technique is light-sheet microscopy, which enables rapid 3D imaging. Our pathology-engineering collaboration has resulted in an open-top light-sheet (OTLS) microscope that is specifically tailored to the needs of pathology practice.To present an image atlas of OTLS images of prostate core needle biopsies.Core needle biopsies (N = 9) were obtained from fresh radical prostatectomy specimens. Each biopsy was fixed in formalin, dehydrated in ethanol, stained with TO-PRO3 and eosin, optically cleared, and imaged using OTLS microscopy. The biopsies were then processed, paraffin embedded, and sectioned. Hematoxylin-eosin and immunohistochemical staining for cytokeratin 5 and cytokeratin 8 was performed.Benign and neoplastic histologic structures showed high fidelity between OTLS and traditional light microscopy. OTLS microscopy had no discernible effect on hematoxylin-eosin or immunohistochemical staining in this pilot study. The 3D histology information obtained using OTLS microscopy enabled new structural insights, including the observation of cribriform and well-formed gland morphologies within the same contiguous glandular structures, as well as the continuity of poorly formed glands with well-formed glands.Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylin-eosin histology images, with the added benefit of useful 3D structural information. Further studies are needed to continue to document the OTLS appearance of a wide range of tissues and to better understand 3D histologic structures.

  View details for DOI 10.5858/arpa.2018-0466-OA

  View details for Web of Science ID 000483384800009

  View details for PubMedID 30892067

  View details for PubMedCentralID PMC7402418

• Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues NATURE COMMUNICATIONS Glaser, A. K., Reder, N. P., Chen, Y., Yin, C., Wei, L., Kang, S., Barner, L. A., Xie, W., McCarty, E. F., Mao, C., Halpern, A. R., Stoltzfus, C. R., Daniels, J. S., Gerner, M. Y., Nicovich, P. R., Vaughan, J. C., True, L. D., Liu, J. T. C. 2019; 10: 2781

  Abstract

  Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

  View details for DOI 10.1038/s41467-019-10534-0

  View details for Web of Science ID 000474235700001

  View details for PubMedID 31273194

  View details for PubMedCentralID PMC6609674

• Toward Quantitative Neurosurgical Guidance With High-Resolution Microscopy of 5-Aminolevulinic Acid-Induced Protoporphyrin IX FRONTIERS IN ONCOLOGY Wei, L., Fujita, Y., Sanai, N., Liu, J. T. C. 2019; 9: 592

  Abstract

  Low-power fluorescence microscopy of 5-ALA-induced PpIX has emerged as a valuable intraoperative imaging technology for improving the resection of malignant gliomas. However, current fluorescence imaging tools are not highly sensitive nor quantitative, which limits their effectiveness for optimizing operative decisions near the surgical margins of gliomas, in particular non-enhancing low-grade gliomas. Intraoperative high-resolution optical-sectioning microscopy can potentially serve as a valuable complement to low-power fluorescence microscopy by providing reproducible quantification of tumor parameters at the infiltrative margins of diffuse gliomas. In this forward-looking perspective article, we provide a brief discussion of recent technical advancements, pilot clinical studies, and our vision of the future adoption of handheld optical-sectioning microscopy at the final stages of glioma surgeries to enhance the extent of resection. We list a number of challenges for clinical acceptance, as well as potential strategies to overcome such obstacles for the surgical implementation of these in vivo microscopy techniques.

  View details for DOI 10.3389/fonc.2019.00592

  View details for Web of Science ID 000473636200001

  View details for PubMedID 31334117

  View details for PubMedCentralID PMC6616084

• Trends and Challenges for the Clinical Adoption of Fluorescence-Guided Surgery JOURNAL OF NUCLEAR MEDICINE Liu, J. T. C., Sanai, N. 2019; 60 (6): 756-757

  View details for DOI 10.2967/jnumed.119.226795

  View details for Web of Science ID 000470084400019

  View details for PubMedID 30877178

  View details for PubMedCentralID PMC6581224

• Label-free <i>in vivo</i> pathology of human epithelia with a high-speed handheld dual-axis confocal microscope JOURNAL OF BIOMEDICAL OPTICS Yin, C., Wei, L., Abeytunge, S., Peterson, G., Rajadhyaksha, M., Liu, J. T. C. 2019; 24 (3): 30501

  Abstract

  There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectance-based handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15  frames/s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1  μm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device.

  View details for DOI 10.1117/1.JBO.24.3.030501

  View details for Web of Science ID 000463886200001

  View details for PubMedID 32717147

  View details for PubMedCentralID PMC6435977

• Rapid pathology of lumpectomy margins with open open-top light-sheet (OTLS) microscopy BIOMEDICAL OPTICS EXPRESS Chen, Y., Xie, W., Glaser, A. K., Reder, N. P., Mao, C., Dintzis, S. M., Vaughan, J. C., Liu, J. T. C. 2019; 10 (3): 1257-1272

  Abstract

  Open-top light-sheet microscopy is a technique that can potentially enable rapid ex vivo inspection of large tissue surfaces and volumes. Here, we have optimized an open-top light-sheet (OTLS) microscope and image-processing workflow for the comprehensive examination of surgical margin surfaces, and have also developed a novel fluorescent analog of H&E staining that is robust for staining fresh unfixed tissues. Our tissue-staining method can be achieved within 2.5 minutes followed by OTLS microscopy of lumpectomy surfaces at a rate of up to 1.5 cm2/minute. An image atlas is presented to show that OTLS image quality surpasses that of intraoperative frozen sectioning and can approximate that of gold-standard H&E histology of formalin-fixed paraffin-embedded (FFPE) tissues. Qualitative evidence indicates that these intraoperative methods do not interfere with downstream post-operative H&E histology and immunohistochemistry. These results should facilitate the translation of OTLS microscopy for intraoperative guidance of lumpectomy and other surgical oncology procedures.

  View details for DOI 10.1364/BOE.10.001257

  View details for Web of Science ID 000460138400016

  View details for PubMedID 30891344

  View details for PubMedCentralID PMC6420271

• Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins JOURNAL OF BIOMEDICAL OPTICS Xie, W., Chen, Y., Wang, Y., Wei, L., Yin, C., Glaser, A. K., Fauver, M. E., Seibel, E. J., Dintzis, S. M., Vaughan, J. C., Reder, N. P., Liu, J. T. C. 2019; 24 (2): 1-11

  Abstract

  Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1  %   of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5  min  /  cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5  min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.

  View details for DOI 10.1117/1.JBO.24.2.026501

  View details for Web of Science ID 000463885200019

  View details for PubMedID 30737911

  View details for PubMedCentralID PMC6368047

• Modeling the binding and diffusion of receptor-targeted nanoparticles topically applied on fresh tissue specimens PHYSICS IN MEDICINE AND BIOLOGY Kang, S., Xu, X., Navarro, E., Wang, Y., Liu, J. T. C., Tichauer, K. M. 2019; 64 (4): 045013

  Abstract

  Nanoparticle (NP) contrast agents targeted to cancer biomarkers are increasingly being engineered for the early detection of cancer, guidance of therapy, and monitoring of response. There have been recent efforts to topically apply biomarker-targeted NPs on tissue surfaces to image the expression of cell-surface receptors over large surface areas as a means of evaluating tumor margins to guide wide local excision surgeries. However, diffusion and nonspecific binding of the NPs present challenges for relating NP retention on the tissue surface with the expression of cancer cell receptors. Paired-agent methods that employ a secondary 'control' NP to account for these nonspecific effects can improve cancer detection. Yet these paired-agent methods introduce multidimensional complexity (with tissue staining, rinsing, imaging, and data analysis protocols all being subject to alteration), and could be greatly simplified with accurate, predictive in silico models of NP binding and diffusion. Here, we outline and validate such a model to predict the diffusion, as well as specific and nonspecific binding, of targeted and control NPs topically applied on tissue surfaces. In order to inform the model, in vitro experiments were performed to determine relevant NP diffusion and binding rate constants in tissues. The predictive capacity of the model was validated by comparing simulated distributions of various sizes of NPs in comparison with experimental results. The regression of predicted and experimentally measured concentration-depth profiles yielded  <15% error (compared to ~70% error obtained using a previous model of NP diffusion and binding).

  View details for DOI 10.1088/1361-6560/aaff81

  View details for Web of Science ID 000459135900001

  View details for PubMedID 30654346

  View details for PubMedCentralID PMC7444409

• Visualization technologies for 5-ALA-based fluorescence-guided surgeries JOURNAL OF NEURO-ONCOLOGY Wei, L., Roberts, D. W., Sanai, N., Liu, J. T. C. 2019; 141 (3): 495-505

  Abstract

  5-ALA-based fluorescence-guided surgery has been shown to be a safe and effective method to improve intraoperative visualization and resection of malignant gliomas. However, it remains ineffective in guiding the resection of lower-grade, non-enhancing, and deep-seated tumors, mainly because these tumors do not produce detectable fluorescence with conventional visualization technologies, namely, wide-field (WF) surgical microscopy.We describe some of the main factors that limit the sensitivity and accuracy of conventional WF surgical microscopy, and then provide a survey of commercial and research prototypes being developed to address these challenges, along with their principles, advantages and disadvantages, as well as the current status of clinical translation for each technology. We also provide a neurosurgical perspective on how these visualization technologies might best be implemented for guiding glioma surgeries in the future.Detection of PpIX expression in low-grade gliomas and at the infiltrative margins of all gliomas has been achieved with high-sensitivity probe-based visualization techniques. Deep-tissue PpIX imaging of up to 5 mm has also been achieved using red-light illumination techniques. Spectroscopic approaches have enabled more accurate quantification of PpIX expression.Advancements in visualization technologies have extended the sensitivity and accuracy of conventional WF surgical microscopy. These technologies will continue to be refined to further improve the extent of resection in glioma patients using 5-ALA-induced fluorescence.

  View details for DOI 10.1007/s11060-018-03077-9

  View details for Web of Science ID 000458256700004

  View details for PubMedID 30554344

  View details for PubMedCentralID PMC7707111

• Handheld line-scanned dual-axis confocal microscope with pistoned MEMS actuation for flat-field fluorescence imaging OPTICS LETTERS Wei, L., Yin, C., Fujita, Y., Sanai, N., Liu, J. T. C. 2019; 44 (3): 671-674

  Abstract

  A handheld line-scanned dual-axis confocal (LS-DAC) microscope has been developed for high-speed (16 frames/s) fluorescence imaging of tissues with sub-nuclear resolution. This is the first miniature fluorescence LS-DAC system that has been fully packaged for handheld clinical use on patients. A novel micro-electro-mechanical system scanning mechanism, with synchronized tilting and pistoning, is used to achieve flat-field en face imaging. We show that this facilitates video mosaicking to generate images that sample an extended lateral field of view.

  View details for DOI 10.1364/OL.44.000671

  View details for Web of Science ID 000457292400053

  View details for PubMedID 30702707

  View details for PubMedCentralID PMC7723749

• Dual-Axis Confocal Microscopy for Point-of-Care Pathology IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS Wei, L., Yin, C., Liu, J. T. C. 2019; 25 (1)

  Abstract

  Dual-axis confocal (DAC) microscopy is an optical imaging modality that utilizes simple low-numerical aperture (NA) lenses to achieve effective optical sectioning and superior image contrast in biological tissues. The unique architecture of DAC microscopy also provides some advantages for miniaturization, facilitating the development of endoscopic and handheld DAC systems for in vivo imaging. This article reviews the principles of DAC microscopy, including its differences from conventional confocal microscopy, and surveys several variations of DAC microscopy that have been developed and investigated as non-invasive real-time alternatives to conventional biopsy and histopathology.

  View details for DOI 10.1109/JSTQE.2018.2854572

  View details for Web of Science ID 000439991500001

  View details for PubMedID 30872909

  View details for PubMedCentralID PMC6411089

• A Raman Imaging Approach Using CD47 Antibody-Labeled SERS Nanoparticles for Identifying Breast Cancer and Its Potential to Guide Surgical Resection. Nanomaterials (Basel, Switzerland) Davis, R. M., Campbell, J. L., Burkitt, S., Qiu, Z., Kang, S., Mehraein, M., Miyasato, D., Salinas, H., Liu, J. T., Zavaleta, C. 2018; 8 (11)

  Abstract

  Raman spectroscopic imaging has shown great promise for improved cancer detection and localization with the use of tumor targeting surface enhanced Raman scattering (SERS) nanoparticles. With the ultrasensitive detection and multiplexing capabilities that SERS imaging has to offer, scientists have been investigating several clinical applications that could benefit from this unique imaging strategy. Recently, there has been a push to develop new image-guidance tools for surgical resection to help surgeons sensitively and specifically identify tumor margins in real time. We hypothesized that SERS nanoparticles (NPs) topically applied to breast cancer resection margins have the potential to provide real-time feedback on the presence of residual cancer in the resection margins during lumpectomy. Here, we explore the ability of SERS nanoparticles conjugated with a cluster of differentiation-47 (CD47) antibody to target breast cancer. CD47 is a cell surface receptor that has recently been shown to be overexpressed on several solid tumor types. The binding potential of our CD47-labeled SERS nanoparticles was assessed using fluorescence assisted cell sorting (FACS) on seven different human breast cancer cell lines, some of which were triple negative (negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2)). Xenograft mouse models were also used to assess the ability of our Raman imaging system to identify tumor from normal tissue. A ratiometric imaging strategy was used to quantify specific vs. nonspecific probe binding, resulting in improved tumor-to-background ratios. FACS analysis showed that CD47-labeled SERS nanoparticles bound to seven different breast cancer cell lines at levels 12-fold to 70-fold higher than isotype control-labeled nanoparticles (p < 0.01), suggesting that our CD47-targeted nanoparticles actively bind to CD47 on breast cancer cells. In a mouse xenograft model of human breast cancer, topical application of CD47-targeted nanoparticles to excised normal and cancer tissue revealed increased binding of CD47-targeted nanoparticles on tumor relative to normal adjacent tissue. The findings of this study support further investigation and suggest that SERS nanoparticles topically applied to breast cancer could guide more complete surgical resection during lumpectomy.

  View details for PubMedID 30463284

• Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging SCIENTIFIC REPORTS Glaser, A. K., Chen, Y., Yin, C., Wei, L., Barner, L. A., Reder, N. P., Liu, J. T. C. 2018; 8: 13878

  Abstract

  Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple and passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides mitigation of shadowing artifacts and contrast-enhanced imaging of fluorescently labeled samples.

  View details for DOI 10.1038/s41598-018-32367-5

  View details for Web of Science ID 000444762700008

  View details for PubMedID 30224740

  View details for PubMedCentralID PMC6141597

• Reaction-Driven Nucleation Theory JOURNAL OF PHYSICAL CHEMISTRY C Wall, M. A., Cossairt, B. M., Liu, J. T. C. 2018; 122 (17): 9671-9679

  View details for DOI 10.1021/acs.jpcc.8b01368

  View details for Web of Science ID 000431723700046

• Microscopic investigation of" topically applied nanoparticles for molecular imaging of fresh tissue surfaces JOURNAL OF BIOPHOTONICS Kang, S., Wang, Y., Xu, X., Navarro, E., Tichauer, K. M., Liu, J. T. C. 2018; 11 (4): e201700246

  Abstract

  Previous studies have shown that functionalized nanoparticles (NPs) topically applied on fresh tissues are able to rapidly target cell-surface protein biomarkers of cancer. Furthermore, studies have shown that a paired-agent approach, in which an untargeted NP is co-administered with a panel of targeted NPs, controls for the nonspecific behavior of the NPs, enabling quantitative imaging of biomarker expression. However, given the complexities in nonspecific accumulation, diffusion, and chemical binding of targeted NPs in tissues, studies are needed to better understand these processes at the microscopic scale. Here, fresh tissues were stained with a paired-agent approach, frozen, and sectioned to image the depth-dependent accumulation of targeted and untargeted NPs. The ratio of targeted-to-untargeted NP concentrations-a parameter used to distinguish between tumor and benign tissues-was found to diminish with increasing NP diffusion depths due to nonspecific accumulation and poor washout. It was then hypothesized and experimentally demonstrated that larger NPs would exhibit less diffusion below tissue surfaces, enabling higher targeted-to-untargeted NP ratios. In summary, these methods and investigations have enabled the design of NP agents with improved sensitivity and contrast for rapid molecular imaging of fresh tissues.

  View details for DOI 10.1002/jbio.201700246

  View details for Web of Science ID 000430178100025

  View details for PubMedID 29227576

  View details for PubMedCentralID PMC5903997

• High-speed Raman-encoded molecular imaging of freshly excised tissue surfaces with topically applied SERRS nanoparticles JOURNAL OF BIOMEDICAL OPTICS Wang, Y., Yang, Q., Kang, S., Wall, M. A., Liu, J. T. C. 2018; 23 (4): 1-8

  Abstract

  Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) are increasingly being engineered for a variety of disease-detection and treatment applications. For example, we have previously developed a fiber-optic Raman-encoded molecular imaging (REMI) system for spectral imaging of biomarker-targeted SERS NPs topically applied on tissue surfaces to identify residual tumors at surgical margins. Although accurate tumor detection was achieved, the commercial SERS NPs used in our previous studies lacked the signal strength to enable high-speed imaging with high pixel counts (large fields of view and/or high spatial resolution), which limits their use for certain time-constrained clinical applications. As a solution, we explored the use of surface-enhanced resonant Raman scattering (SERRS) NPs to enhance imaging speeds. The SERRS NPs were synthesized de novo, and then conjugated to HER2 antibodies to achieve high binding affinity, as validated by flow cytometry. Under identical tissue-staining and imaging conditions, the targeted SERRS NPs enabled reliable identification of HER2-overexpressed tumor xenografts with 50-fold-enhanced imaging speed compared with our standard targeted SERS NPs. This enables our REMI system to image tissue surfaces at a rate of 150  cm2 per minute at a spatial resolution of 0.5 mm.

  View details for DOI 10.1117/1.JBO.23.4.046005

  View details for Web of Science ID 000435415700007

  View details for PubMedID 29658229

  View details for PubMedCentralID PMC5899991

• Identification of Tissue Invasive Fungi by 2D and 3D Light Sheet Microscopy Lieberman, J., Glaser, A. K., Reder, N., True, L. D., Bryan, A., Liu, J. T., Bourassa, L. NATURE PUBLISHING GROUP. 2018: 579

  View details for Web of Science ID 000459341002480

• Thick tissue diffusion model with binding to optimize topical staining in fluorescence breast cancer margin imaging Xu, X., Kang, S., Navarro-Comes, E., Wang, Y., Liu, J. T. C., Tichauer, K. M. edited by Pogue, B. W., Gioux, S. SPIE-INT SOC OPTICAL ENGINEERING. 2018

  View details for DOI 10.1117/12.2286538

  View details for Web of Science ID 000451703500005

• A handheld MEMS-scanned in vivo optical-sectioning microscope for early detection and surgical guidance Yin, C., Wei, L., Abeytunge, S., Peterson, G., Glaser, A. K., Mandella, M. J., Rajadhyaksha, M., Liu, J. T. C. edited by Winzer, P., Tsang, H. K., Capmany, J., Yao, J., Fontaine, N., Dutta, N. IEEE. 2018

  View details for Web of Science ID 000460542800066

• MEASURING AND MITIGATING SPECKLE NOISE IN DUAL-AXIS CONFOCAL MICROSCOPY IMAGES Gigilashvili, D., Yin, C., Liu, J. T. C., Hardeberg, J., Pedersen, M. edited by DiBaja, G. S., Gallo, L., Yetongnon, K., Dipanda, A., CastrillonSantana, M., Chbeir, R. IEEE. 2018: 281-288

  View details for DOI 10.1109/SITIS.2018.00050

  View details for Web of Science ID 000469258400039

• Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy CANCER RESEARCH Wang, Y., Reder, N. P., Kang, S., Glaser, A. K., Yang, Q., Wall, M. A., Javid, S. H., Dintzis, S. M., Liu, J. T. C. 2017; 77 (16): 4506-4516

  Abstract

  Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.

  View details for DOI 10.1158/0008-5472.CAN-17-0709

  View details for Web of Science ID 000407613500027

  View details for PubMedID 28615226

  View details for PubMedCentralID PMC5559330

• Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens NATURE BIOMEDICAL ENGINEERING Glaser, A. K., Reder, N. P., Chen, Y., McCarty, E. F., Yin, C., Wei, L., Wang, Y., True, L. D., Liu, J. T. C. 2017; 1 (7)

  Abstract

  For the 1.7 million patients per year in the U.S. who receive a new cancer diagnosis, treatment decisions are largely made after a histopathology exam. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 μm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 sec/cm2), and volumetric assessment of optically cleared core-needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens.

  View details for DOI 10.1038/s41551-017-0084

  View details for Web of Science ID 000418857900001

  View details for PubMedID 29750130

  View details for PubMedCentralID PMC5940348

• Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues PHYSICS IN MEDICINE AND BIOLOGY Xu, X., Wang, Y., Xiang, J., Liu, J. T. C., Tichauer, K. M. 2017; 62 (12): 5098-5113

  Abstract

  Conventional molecular assessment of tissue through histology, if adapted to fresh thicker samples, has the potential to enhance cancer detection in surgical margins and monitoring of 3D cell culture molecular environments. However, in thicker samples, substantial background staining is common despite repeated rinsing, which can significantly reduce image contrast. Recently, 'paired-agent' methods-which employ co-administration of a control (untargeted) imaging agent-have been applied to thick-sample staining applications to account for background staining. To date, these methods have included (1) a simple ratiometric method that is relatively insensitive to noise in the data but has accuracy that is dependent on the staining protocol and the characteristics of the sample; and (2) a complex paired-agent kinetic modeling method that is more accurate but is more noise-sensitive and requires a precise serial rinsing protocol. Here, a new simplified mathematical model-the rinsing paired-agent model (RPAM)-is derived and tested that offers a good balance between the previous models, is adaptable to arbitrary rinsing-imaging protocols, and does not require calibration of the imaging system. RPAM is evaluated against previous models and is validated by comparison to estimated concentrations of targeted biomarkers on the surface of 3D cell culture and tumor xenograft models. This work supports the use of RPAM as a preferable model to quantitatively analyze targeted biomarker concentrations in topically stained thick tissues, as it was found to match the accuracy of the complex paired-agent kinetic model while retaining the low noise-sensitivity characteristics of the ratiometric method.

  View details for DOI 10.1088/1361-6560/aa6cf1

  View details for Web of Science ID 000402376700001

  View details for PubMedID 28548970

  View details for PubMedCentralID PMC5817640

• Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology JOURNAL OF BIOMEDICAL OPTICS Wei, L., Chen, Y., Yin, C., Borwege, S., Sanai, N., Liu, J. T. C. 2017; 22 (4): 46005

  Abstract

  Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

  View details for DOI 10.1117/1.JBO.22.4.046005

  View details for Web of Science ID 000400896100036

  View details for PubMedID 28418534

  View details for PubMedCentralID PMC5390779

• Clarification of Prostate Core Needle Biopsies with 2,2′ Thiodiethanol Rapidly and Efficiently Prepares Tissue for 3D Light-Sheet Microscopy McCarty, E., Reder, N. P., Glaser, A. K., Chen, Y., Liu, J. T. C., True, L. NATURE PUBLISHING GROUP. 2017: 532A

  View details for Web of Science ID 000394467302708

• 3D Light-Sheet Microscopy Improves the Accuracy of Grading Prostate Cancer by Distinguishing Pattern 3 Glands from Poorly Formed Pattern 4 Glands Reder, N. P., Glaser, A. K., McCarty, E., Chen, Y., Liu, J. T. C., True, L. NATURE PUBLISHING GROUP. 2017: 251A

  View details for Web of Science ID 000393724401288

• Cost-Effective Triaging of Prostatectomy Specimens Using Light-Sheet Microscopy Reder, N. P., Glaser, A. K., McCarty, E., Chen, Y., Liu, J. T. C., True, L. NATURE PUBLISHING GROUP. 2017: 517A

  View details for Web of Science ID 000394467302651

• A handheld MEMS-based line-scanned dual-axis confocal microscope for early cancer detection and surgical guidance (Conference Presentation) Chen, Y., Yin, C., Wei, L., Glaser, A. K., Abeytunge, S., Peterson, G., Mandella, M. J., Sanai, N., Rajadhyaksha, M., Liu, J. T. edited by Tearney, G. J., Wang, T. D. SPIE-INT SOC OPTICAL ENGINEERING. 2017

  View details for DOI 10.1117/12.2250440

  View details for Web of Science ID 000401132400020

• Numerical modeling of illumination and detection methods for light-sheet microscopy of optically clear biological tissues Glaser, A. K., Liu, J. T. C., IEEE Photon Soc IEEE. 2017: 351-352

  View details for Web of Science ID 000426792600154

• Multiplexed Optical Imaging of Tumor-Directed Nanoparticles: A Review of Imaging Systems and Approaches. Nanotheranostics Wang, Y. W., Reder, N. P., Kang, S., Glaser, A. K., Liu, J. T. 2017; 1 (4): 369-388

  Abstract

  In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.

  View details for DOI 10.7150/ntno.21136

  View details for PubMedID 29071200

• Bessel-beam illumination in dual-axis confocal microscopy mitigates resolution degradation caused by refractive heterogeneities JOURNAL OF BIOPHOTONICS Chen, Y., Glaser, A., Liu, J. T. C. 2017; 10 (1): 68-74

  Abstract

  One of the main challenges for laser-scanning microscopy of biological tissues with refractive heterogeneities is the degradation in spatial resolution that occurs as a result of beam steering and distortion. This challenge is particularly significant for dual-axis confocal (DAC) microscopy, which achieves improved spatial-filtering and optical-sectioning performance over traditional confocal microscopy through off-axis illumination and collection of light with low-numerical aperture (NA) beams that must intersect precisely at their foci within tissues. DAC microscope image quality is sensitive to positional changes and distortions of these illumination- and collection-beam foci. Previous studies have shown that Bessel beams display improved positional stability and beam quality than Gaussian beams when propagating through tissues with refractive heterogeneities, which suggests that Bessel-beam illumination may enhance DAC microscopy of such tissues. Here, we utilize both Gaussian and Bessel illumination in a point-scanned DAC microscope and quantify the resultant degradation in resolution when imaging within heterogeneous optical phantoms and fresh tissues. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination.

  View details for DOI 10.1002/jbio.201600196

  View details for Web of Science ID 000394849400006

  View details for PubMedID 27667127

  View details for PubMedCentralID PMC5243863

• Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection-Enhanced Topical Staining with SERS-Coded Nanoparticles SMALL Wang, Y. W., Doerksen, J. D., Kang, S., Walsh, D., Yang, Q., Hong, D., Liu, J. T. C. 2016; 12 (40): 5612-5621

  Abstract

  There is a need for intraoperative imaging technologies to guide breast-conserving surgeries and to reduce the high rates of re-excision for patients in which residual tumor is found at the surgical margins during postoperative pathology analyses. Feasibility studies have shown that utilizing topically applied surface-enhanced Raman scattering (SERS) nanoparticles (NPs), in conjunction with the ratiometric imaging of targeted versus untargeted NPs, enables the rapid visualization of multiple cell-surface biomarkers of cancer that are overexpressed at the surfaces of freshly excised breast tissues. In order to reliably and rapidly perform multiplexed Raman-encoded molecular imaging of large numbers of biomarkers (with five or more NP flavors), an enhanced staining method has been developed in which tissue surfaces are cyclically dipped into an NP-staining solution and subjected to high-frequency mechanical vibration. This dipping and mechanical vibration (DMV) method promotes the convection of the SERS NPs at fresh tissue surfaces, which accelerates their binding to their respective biomarker targets. By utilizing a custom-developed device for automated DMV staining, this study demonstrates the ability to simultaneously image four cell-surface biomarkers of cancer at the surfaces of fresh human breast tissues with a mixture of five flavors of SERS NPs (four targeted and one untargeted control) topically applied for 5 min and imaged at a spatial resolution of 0.5 mm and a raster-scanned imaging rate of >5 cm2 min-1 .

  View details for DOI 10.1002/smll.201601829

  View details for Web of Science ID 000389401300012

  View details for PubMedID 27571395

  View details for PubMedCentralID PMC5462459

• Modulated-Alignment Dual-Axis (MAD) Confocal Microscopy Optimized for Speed and Contrast IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING Leigh, S. Y., Chen, Y., Liu, J. T. C. 2016; 63 (10): 2119-2124

  Abstract

  Modulated-alignment dual-axis (MAD) confocal microscopy combines the benefits of dual-axis confocal (DAC) microscopy and focal-modulation microscopy (FMM) for rejecting out-of-focus and multiply scattered light in tissues. The DAC architecture, which utilizes off-axis and separated beam paths for illumination and detection, has previously been shown to be superior to single-axis confocal (SAC) microscopy for the spatial filtering (rejection) of unwanted background light. With the MAD approach, a modulation of the alignment between the illumination and collection beam paths tags ballistic photons emanating from the focal volume with a characteristic radio frequency that can be extracted and separated from background signal using lock-in detection. We report here an optimized form of MAD confocal microscopy where we have fully mitigated tradeoffs in performance in an initial proof-of-concept system in order to recover the imaging speed of DAC microscopy while retaining contrast enhancement of 6 dB (signal-to-background ratio) with a secondary improvement in optical-sectioning and in-plane resolution. Validation is demonstrated with light-scattering tissue phantoms and freshly excised tissues.

  View details for DOI 10.1109/TBME.2015.2511581

  View details for Web of Science ID 000384570200015

  View details for PubMedID 28055837

  View details for PubMedCentralID PMC5047853

• Multiplexed Molecular Imaging of Biomarker-Targeted SERS Nanoparticles on Fresh Tissue Specimens with Channel-Compressed Spectrometry PLOS ONE Kang, S., Wang, Y., Reder, N. P., Liu, J. T. C. 2016; 11 (9): e0163473

  Abstract

  Biomarker-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have been explored as a viable option for targeting and imaging multiple cell-surface protein biomarkers of cancer. While it has been demonstrated that this Raman-encoded molecular imaging (REMI) technology may potentially be used to guide tumor-resection procedures, the REMI strategy would benefit from further improvements in imaging speed. Previous implementations of REMI have utilized 1024 spectral channels (camera pixels) in a commercial spectroscopic CCD to detect the spectral signals from multiplexed SERS NPs, a strategy that enables accurate demultiplexing of the relative concentration of each NP "flavor" within a mixture. Here, we investigate the ability to significantly reduce the number of spectral-collection channels while maintaining accurate imaging and demultiplexing of up to five SERS NP flavors, a strategy that offers the potential for improved imaging speed and/or detection sensitivity in future systems. This strategy was optimized by analyzing the linearity of five multiplexed flavors of SERS NPs topically applied on tissues. The accuracy of this binning approach was then validated by staining tumor xenografts and human breast tumor specimens with a mixture of five NP flavors (four targeted NPs and one untargeted NP) and performing ratiometric imaging of specific vs. nonspecific NP accumulation. We demonstrate that with channel-compressed spectrometry using as few as 16 channels, it is possible to perform REMI with five NP flavors, with < 20% error, at low concentrations (< 10 pM) that are relevant for clinical applications.

  View details for DOI 10.1371/journal.pone.0163473

  View details for Web of Science ID 000384328500060

  View details for PubMedID 27685991

  View details for PubMedCentralID PMC5042405

• Fractal propagation method enables realistic optical microscopy simulations in biological tissues OPTICA Glaser, A. K., Chen, Y., Liu, J. T. C. 2016; 3 (8): 861-869

  Abstract

  Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method's flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy.

  View details for DOI 10.1364/OPTICA.3.000861

  View details for Web of Science ID 000382007700013

  View details for PubMedID 28983499

  View details for PubMedCentralID PMC5626453

• Surgical Guidance via Multiplexed Molecular Imaging of Fresh Tissues Labeled with SERS-Coded Nanoparticles. IEEE journal of selected topics in quantum electronics : a publication of the IEEE Lasers and Electro-optics Society Wang, Y., Kang, S., Doerksen, J. D., Glaser, A. K., Liu, J. T. 2016; 22 (4)

  Abstract

  The imaging of dysregulated cell-surface receptors (or biomarkers) is a potential means of identifying the presence of cancer with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Recently, surface-enhanced Raman-scattering (SERS) nanoparticles (NPs) have attracted wide interest due to their potential for sensitive and multiplexed biomarker detection. In this review, we focus on the most recent advances in tumor imaging using SERS-coded NPs. A brief introduction of the structure and optical properties of SERS NPs is provided, followed by a detailed discussion of key imaging issues such as the administration of NPs in tissue (topical versus systemic), the optical configuration and imaging approach of Raman imaging systems, spectral demultiplexing methods for quantifying NP concentrations, and the disambiguation of specific vs. nonspecific sources of contrast through ratiometric imaging of targeted and untargeted (control) NP pairs. Finally, future challenges and directions are briefly outlined.

  View details for PubMedID 27524875

• Detection and delineation of oral cancer with a PARP1 targeted optical imaging agent SCIENTIFIC REPORTS Kossatz, S., Brand, C., Gutiontov, S., Liu, J. T. C., Lee, N. Y., Goenen, M., Weber, W. A., Reiner, T. 2016; 6: 21371

  Abstract

  Earlier and more accurate detection of oral squamous cell carcinoma (OSCC) is essential to improve the prognosis of patients and to reduce the morbidity of surgical therapy. Here, we demonstrate that the nuclear enzyme Poly(ADP-ribose)Polymerase 1 (PARP1) is a promising target for optical imaging of OSCC with the fluorescent dye PARPi-FL. In patient-derived OSCC specimens, PARP1 expression was increased 7.8 ± 2.6-fold when compared to normal tissue. Intravenous injection of PARPi-FL allowed for high contrast in vivo imaging of human OSCC models in mice with a surgical fluorescence stereoscope and high-resolution imaging systems. The emitted signal was specific for PARP1 expression and, most importantly, PARPi-FL can be used as a topical imaging agent, spatially resolving the orthotopic tongue tumors in vivo. Collectively, our results suggest that PARP1 imaging with PARPi-FL can enhance the detection of oral cancer, serve as a screening tool and help to guide surgical resections.

  View details for DOI 10.1038/srep21371

  View details for Web of Science ID 000370493600001

  View details for PubMedID 26900125

  View details for PubMedCentralID PMC4761964

• Quantitative molecular phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy SCIENTIFIC REPORTS Wang, Y., Kang, S., Khan, A., Ruttner, G., Leigh, S. Y., Murray, M., Abeytunge, S., Peterson, G., Rajadhyaksha, M., Dintzis, S., Javid, S., Liu, J. T. C. 2016; 6: 21242

  Abstract

  There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.

  View details for DOI 10.1038/srep21242

  View details for Web of Science ID 000370226500001

  View details for PubMedID 26878888

  View details for PubMedCentralID PMC4754709

• Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology. Biomedical optics express Yin, C., Glaser, A. K., Leigh, S. Y., Chen, Y., Wei, L., Pillai, P. C., Rosenberg, M. C., Abeytunge, S., Peterson, G., Glazowski, C., Sanai, N., Mandella, M. J., Rajadhyaksha, M., Liu, J. T. 2016; 7 (2): 251-63

  Abstract

  There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device.

  View details for DOI 10.1364/BOE.7.000251

  View details for PubMedID 26977337

  View details for PubMedCentralID PMC4771446

• Bessel beam illumination reduces resolution degradation due to micro-architectural heterogeneities for dual-axis confocal microscopy of tissues Chen, Y., Liu, J. T. C., IEEE IEEE. 2016: 148-149

  View details for Web of Science ID 000406880100078

• Video-rate <i>in vivo</i> fluorescence imaging with a line-scanned dual-axis confocal microscope JOURNAL OF BIOMEDICAL OPTICS Chen, Y., Wang, D., Khan, A., Wang, Y., Borwege, S., Sanai, N., Liu, J. T. C. 2015; 20 (10): 106011

  Abstract

  Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.

  View details for DOI 10.1117/1.JBO.20.10.106011

  View details for Web of Science ID 000366017100032

  View details for PubMedID 26509413

  View details for PubMedCentralID PMC4881331

• <i>In vivo</i> multiplexed molecular imaging of esophageal cancer via spectral endoscopy of topically applied SERS nanoparticles BIOMEDICAL OPTICS EXPRESS Wang, Y., Kang, S., Khan, A., Bao, P. Q., Liu, J. T. C. 2015; 6 (10): 3714-3723

  Abstract

  The biological investigation and detection of esophageal cancers could be facilitated with an endoscopic technology to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and investigation through the sensitive and multiplexed detection of cell-surface biomarkers. Here, we demonstrate that the topical application and endoscopic imaging of a multiplexed cocktail of receptor-targeted SERS NPs enables the rapid detection of tumors in an orthotopic rat model of esophageal cancer. Antibody-conjugated SERS NPs were topically applied on the lumenal surface of the rat esophagus to target EGFR and HER2, and a miniature spectral endoscope featuring rotational scanning and axial pull-back was employed to comprehensively image the NPs bound on the lumen of the esophagus. Ratiometric analyses of specific vs. nonspecific binding enabled the visualization of tumor locations and the quantification of biomarker expression in agreement with immunohistochemistry and flow cytometry validation data.

  View details for DOI 10.1364/BOE.6.003714

  View details for Web of Science ID 000362236600005

  View details for PubMedID 26504623

  View details for PubMedCentralID PMC4605032

• Quantitative <i>in vivo</i> cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging PHYSICS IN MEDICINE AND BIOLOGY Tichauer, K. M., Wang, Y., Pogue, B. W., Liu, J. T. C. 2015; 60 (14): R239-R269

  Abstract

  The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche.

  View details for DOI 10.1088/0031-9155/60/14/R239

  View details for Web of Science ID 000357620400001

  View details for PubMedID 26134619

  View details for PubMedCentralID PMC4522156

• Feasibility of intraoperative fluorescence imaging of squamous cell carcinoma of the oral cavity using an optical PARP1 inhibitor Kossatz, S., Irwin, C., Leigh, S., Wang, D., Liu, J., Weber, W., Reiner, T. SOC NUCLEAR MEDICINE INC. 2015

  View details for Web of Science ID 000358738800063

• A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles PLOS ONE Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H. 2015; 10 (4)

  Abstract

  The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.

  View details for DOI 10.1371/journal.pone.0123185

  View details for Web of Science ID 000353711600032

  View details for PubMedID 25923788

  View details for PubMedCentralID PMC4414592

• Comparing High-Resolution Microscopy Techniques for Potential Intraoperative Use in Guiding Low-Grade Glioma Resections LASERS IN SURGERY AND MEDICINE Meza, D., Wang, D., Wang, Y., Borwege, S., Sanai, N., Liu, J. T. C. 2015; 47 (4): 289-295

  Abstract

  Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy.Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality).HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast.Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning.

  View details for DOI 10.1002/lsm.22347

  View details for Web of Science ID 000353233100002

  View details for PubMedID 25872487

  View details for PubMedCentralID PMC4500631

• Characterizing the beam steering and distortion of Gaussian and Bessel beams focused in tissues with microscopic heterogeneities BIOMEDICAL OPTICS EXPRESS Chen, Y., Liu, J. T. C. 2015; 6 (4): 1318-1330

  Abstract

  Bessel beams have recently been investigated as a means of improving deep-tissue microscopy in highly scattering and heterogeneous media. It has been suggested that the long depth-of-field and self-reconstructing property of a Bessel beam enables an increased penetration depth of the focused beam in tissues compared to a conventional Gaussian beam. However, a study is needed to better quantify the magnitude of the beam steering as well as the distortion of focused Gaussian and Bessel beams in tissues with microscopic heterogeneities. Here, we have developed an imaging method and quantitative metrics to evaluate the motion and distortion of low-numerical-aperture (NA) Gaussian and Bessel beams focused in water, heterogeneous phantoms, and fresh mouse esophagus tissues. Our results indicate that low-NA Bessel beams exhibit reduced beam-steering artifacts and distortions compared to Gaussian beams, and are therefore potentially useful for microscopy applications in which pointing accuracy and beam quality are critical, such as dual-axis confocal (DAC) microscopy.

  View details for DOI 10.1364/BOE.6.001318

  View details for Web of Science ID 000352228400019

  View details for PubMedID 25909015

  View details for PubMedCentralID PMC4399670

• Quantification of the binding potential of cell-surface receptors in fresh excised specimens via dual-probe modeling of SERS nanoparticles SCIENTIFIC REPORTS Sinha, L., Wang, Y., Yang, C., Khan, A., Brankov, J. G., Liu, J. T. C., Tichauer, K. M. 2015; 5: 8582

  Abstract

  The complete removal of cancerous tissue is a central aim of surgical oncology, but is difficult to achieve in certain cases, especially when the removal of surrounding normal tissues must be minimized. Therefore, when post-operative pathology identifies residual tumor at the surgical margins, re-excision surgeries are often necessary. An intraoperative approach for tumor-margin assessment, insensitive to nonspecific sources of molecular probe accumulation and contrast, is presented employing kinetic-modeling analysis of dual-probe staining using surface-enhanced Raman scattering nanoparticles (SERS NPs). Human glioma (U251) and epidermoid (A431) tumors were implanted subcutaneously in six athymic mice. Fresh resected tissues were stained with an equimolar mixture of epidermal growth factor receptor (EGFR)-targeted and untargeted SERS NPs. The binding potential (BP; proportional to receptor concentration) of EGFR - a cell-surface receptor associated with cancer - was estimated from kinetic modeling of targeted and untargeted NP concentrations in response to serial rinsing. EGFR BPs in healthy, U251, and A431 tissues were 0.06 ± 0.14, 1.13 ± 0.40, and 2.23 ± 0.86, respectively, which agree with flow-cytometry measurements and published reports. The ability of this approach to quantify the BP of cell-surface biomarkers in fresh tissues opens up an accurate new approach to analyze tumor margins intraoperatively.

  View details for DOI 10.1038/srep08582

  View details for Web of Science ID 000349964200002

  View details for PubMedID 25716578

  View details for PubMedCentralID PMC4341215

• Ex-vivo tissue classification of cell surface receptor concentrations using kinetic modeling Sinha, L., Wang, Y., Yang, C., Khan, A., Liu, J. T., Tichauer, K. M. edited by Pogue, B. W., Gioux, S. SPIE-INT SOC OPTICAL ENGINEERING. 2015

  View details for DOI 10.1117/12.2077738

  View details for Web of Science ID 000353556000013

• Comprehensive spectral endoscopy of topically applied SERS nanoparticles in the rat esophagus BIOMEDICAL OPTICS EXPRESS Wang, Y., Khan, A., Leigh, S. Y., Wang, D., Chen, Y., Meza, D., Liu, J. T. C. 2014; 5 (9): 2883-2895

  Abstract

  The early detection and biological investigation of esophageal cancer would benefit from the development of advanced imaging techniques to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and the investigation of cancer progression through the sensitive and multiplexed phenotyping of cell-surface biomarkers. Here, a miniature endoscope featuring rotational scanning and axial pull back has been developed for 2D spectral imaging of SERS NPs topically applied on the lumenal surface of the rat esophagus. Raman signals from low-pM concentrations of SERS NP mixtures are demultiplexed in real time to accurately calculate the concentration and ratio of the NPs. Ex vivo and in vivo experiments demonstrate the feasibility of topical application and imaging of multiplexed SERS NPs along the entire length of the rat esophagus.

  View details for DOI 10.1364/BOE.5.002883

  View details for Web of Science ID 000341650900002

  View details for PubMedID 25401005

  View details for PubMedCentralID PMC4230873

• Trends in Fluorescence Image-Guided Surgery for Gliomas NEUROSURGERY Liu, J. T. C., Meza, D., Sanai, N. 2014; 75 (1): 61-71

  Abstract

  Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low-grade and high-grade glioma patients. However, radiographically complete resections are not often achieved in many cases because of the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins. Here, we provide a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care.

  View details for DOI 10.1227/NEU.0000000000000344

  View details for Web of Science ID 000337725100016

  View details for PubMedID 24618801

  View details for PubMedCentralID PMC4062574

• Modulated-alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues BIOMEDICAL OPTICS EXPRESS Leigh, S. Y., Chen, Y., Liu, J. T. C. 2014; 5 (6): 1709-1720

  Abstract

  A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively 'labels' in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

  View details for DOI 10.1364/BOE.5.001709

  View details for Web of Science ID 000337503900002

  View details for PubMedID 24940534

  View details for PubMedCentralID PMC4052905

• Rapid ratiometric biomarker detection with topically applied SERS nanoparticles TECHNOLOGY Wang, Y., Khan, A., Som, M., Wang, D., Chen, Y., Leigh, S. Y., Meza, D., McVeigh, P. Z., Wilson, B. C., Liu, J. T. C. 2014; 2 (2): 118-132

  Abstract

  Multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) offer the potential for rapid molecular phenotyping of tissues, thereby enabling accurate disease detection as well as patient stratification to guide personalized therapies or to monitor treatment outcomes. The clinical success of molecular diagnostics based on SERS NPs would be facilitated by the ability to accurately identify tissue biomarkers under time-constrained staining and detection conditions with a portable device. In vitro, ex vivo and in vivo experiments were performed to optimize the technology and protocols for the rapid detection (0.1-s integration time) of multiple cell-surface biomarkers with a miniature fiber-optic spectral-detection probe following a brief (5 min) topical application of SERS NPs on tissues. Furthermore, we demonstrate that the simultaneous detection and ratiometric quantification of targeted and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to nonspecific variations in NP concentrations.

  View details for DOI 10.1142/S2339547814500125

  View details for Web of Science ID 000216886500004

  View details for PubMedID 25045721

  View details for PubMedCentralID PMC4103661

• Modulated Alignment Dual-Axis (MAD) Confocal Microscopy for Deep Optical Sectioining in Tissues Leigh, S. Y., Chen, Y., Liu, J. T. C., IEEE IEEE. 2014: 146-148

  View details for DOI 10.1109/ISOT.2014.43

  View details for Web of Science ID 000393497300035

• Rapid multiplexed imaging of cell-surface cancer biomarkers in fresh tissues with targeted SERS nanoparticles Wang, Y., Khan, A., Leigh, S. Y., Liu, J. T. C., IEEE IEEE. 2014: 149-152

  View details for DOI 10.1109/ISOT.2014.44

  View details for Web of Science ID 000393497300036

• Modulated alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues Leigh, S. Y., Chen, Y., Liu, J. T. C. edited by Tkaczyk, T. S. SPIE-INT SOC OPTICAL ENGINEERING. 2014

  View details for DOI 10.1117/12.2057734

  View details for Web of Science ID 000343124000007

• Development and optimization of a line-scanned dual-axis confocal (LS-DAC) microscope for high-speed pathology Wang, D., Chen, Y., Meza, D., Wang, Y., Liu, J. T. C. edited by Tkaczyk, T. S. SPIE-INT SOC OPTICAL ENGINEERING. 2014

  View details for DOI 10.1117/12.2057743

  View details for Web of Science ID 000343124000008

• Rapid multiplexed molecular phenotyping of <i>ex vivo</i> and <i>in vivo</i> tissues with targeted SERS NPs Wang, Y., Khan, A., Som, M., Leigh, S. Y., Wang, D., Chen, Y., McVeigh, P., Wilson, B. C., Liu, J. T. C. edited by Tkaczyk, T. S. SPIE-INT SOC OPTICAL ENGINEERING. 2014

  View details for DOI 10.1117/12.2057716

  View details for Web of Science ID 000343124000006

• A Raman-based endoscopic strategy for multiplexed molecular imaging. Proceedings of the National Academy of Sciences of the United States of America Zavaleta, C. L., Garai, E., Liu, J. T., Sensarn, S., Mandella, M. J., Van de Sompel, D., Friedland, S., Van Dam, J., Contag, C. H., Gambhir, S. S. 2013; 110 (25): E2288-97

  Abstract

  Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

  View details for DOI 10.1073/pnas.1211309110

  View details for PubMedID 23703909

  View details for PubMedCentralID PMC3690865

• Optimizing the performance of dual-axis confocal microscopes via Monte-Carlo scattering simulations and diffraction theory JOURNAL OF BIOMEDICAL OPTICS Chen, Y., Liu, J. T. C. 2013; 18 (6): 066006

  Abstract

  Dual-axis confocal (DAC) microscopy has been found to exhibit superior rejection of out-of-focus and multiply scattered background light compared to conventional single-axis confocal microscopy. DAC microscopes rely on the use of separated illumination and collection beam paths that focus and intersect at a single focal volume (voxel) within tissue. While it is generally recognized that the resolution and contrast of a DAC microscope depends on both the crossing angle of the DAC beams, 2θ, and the focusing numerical aperture of the individual beams, α, a detailed study to investigate these dependencies has not been performed. Contrast and resolution are considered as two main criteria to assess the performance of a point-scanned DAC microscope (DAC-PS) and a line-scanned DAC microscope (DAC-LS) as a function of θ and α. The contrast and resolution of these designs are evaluated by Monte-Carlo scattering simulations and diffraction theory calculations, respectively. These results can be used for guiding the optimal designs of DAC-PS and DAC-LS microscopes.

  View details for DOI 10.1117/1.JBO.18.6.066006

  View details for Web of Science ID 000322341100048

  View details for PubMedID 23733022

  View details for PubMedCentralID PMC3670619

• Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles PLOS ONE Leigh, S. Y., Som, M., Liu, J. T. C. 2013; 8 (4): e62084

  Abstract

  Surface-enhanced Raman scattering (SERS) nanoparticles have been engineered to generate unique fingerprint spectra and are potentially useful as bright contrast agents for molecular diagnostics. One promising strategy for biomedical diagnostics and imaging is to functionalize various particle types ("flavors"), each emitting a unique spectral signature, to target a large multiplexed panel of molecular biomarkers. While SERS particles emit narrow spectral features that allow them to be easily separable under ideal conditions, the presence of competing noise sources and background signals such as detector noise, laser background, and autofluorescence confounds the reliability of demultiplexing algorithms. Results obtained during time-constrained in vivo imaging experiments may not be reproducible or accurate. Therefore, our goal is to provide experimentalists with a metric that may be monitored to enforce a desired bound on accuracy within a user-defined confidence level. We have defined a spectral reliability index (SRI), based on the output of a direct classical least-squares (DCLS) demultiplexing routine, which provides a measure of the reliability of the computed nanoparticle concentrations and ratios. We present simulations and experiments to demonstrate the feasibility of this strategy, which can potentially be utilized for a range of instruments and biomedical applications involving multiplexed SERS nanoparticles.

  View details for DOI 10.1371/journal.pone.0062084

  View details for Web of Science ID 000317909500086

  View details for PubMedID 23620806

  View details for PubMedCentralID PMC3631148

• Real-time pathology through in vivo microscopy. Studies in health technology and informatics Liu, J. T., Loewke, N. O., Mandella, M. J., Leigh, S. Y., Levenson, R. M., Crawford, J. M., Contag, C. H. 2013; 185: 235-264

  Abstract

  Miniature microscopes are being developed to examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to a shift from the current diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to one of non-invasive point-of-care diagnosis with the possibility of treatment in the same session. This potential revolution in disease management may have a major impact on the training of future physicians to include the use and interpretation of real-time in vivo microscopic data, and will also affect the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires multidisciplinary communication and collaboration among clinicians, engineers and scientists. As such, our aim is to provide a forward-looking view of the critical issues facing the development of new technologies and directing clinical education. Here, we focus on the use of in vivo microscopy for detection of malignant and pre-malignant lesions as well as for guiding therapy. We will highlight some of the areas in which in vivo microscopy could address unmet clinical needs, and then review the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become an effective clinical tool.

  View details for PubMedID 23542938

• Microscopic Delineation of Medulloblastoma Margins in a Transgenic Mouse Model Using a Topically Applied VEGFR-1 Probe TRANSLATIONAL ONCOLOGY Wang, D., Chen, Y., Leigh, S. Y., Haeberle, H., Contag, C. H., Liu, J. T. 2012; 5 (6): 408-414

  Abstract

  The unambiguous demarcation of tumor margins is critical at the final stages in the surgical treatment of brain tumors because patient outcomes have been shown to correlate with the extent of resection. Real-time high-resolution imaging with the aid of a tumor-targeting fluorescent contrast agent has the potential to enable intraoperative differentiation of tumor versus normal tissues with accuracy approaching the current gold standard of histopathology. In this study, a monoclonal antibody targeting the vascular endothelial growth factor receptor 1 (VEGFR-1) was conjugated to fluorophores and evaluated as a tumor contrast agent in a transgenic mouse model of medulloblastoma. The probe was administered topically, and its efficacy as an imaging agent was evaluated in vitro using flow cytometry, as well as ex vivo on fixed and fresh tissues through immunohistochemistry and dual-axis confocal microscopy, respectively. Results show a preferential binding to tumor versus normal tissue, suggesting that a topically applied VEGFR-1 probe can potentially be used with real-time intraoperative optical sectioning microscopy to guide brain tumor resections.

  View details for DOI 10.1593/tlo.12277

  View details for Web of Science ID 000313359800003

  View details for PubMedID 23323155

  View details for PubMedCentralID PMC3542836

• A liquid optical phantom with tissue-like heterogeneities for confocal microscopy. Biomedical optics express Wang, D., Chen, Y., Liu, J. T. 2012; 3 (12): 3153-60

  Abstract

  Phantoms play an important role in the development, standardization, and calibration of biomedical imaging devices in laboratory and clinical settings, serving as standards to assess the performance of such devices. Here we present the design of a liquid optical phantom to facilitate the assessment of optical-sectioning microscopes that are being developed to enable point-of-care pathology. This phantom, composed of silica microbeads in an Intralipid base, is specifically designed to characterize a reflectance-based dual-axis confocal (DAC) microscope for skin imaging. The phantom mimics the scattering properties of normal human epithelial tissue in terms of an effective scattering coefficient and a depth-dependent degradation in spatial resolution due to beam steering caused by tissue micro-architectural heterogeneities.

  View details for DOI 10.1364/BOE.3.003153

  View details for PubMedID 23243566

• Assessing the tissue-imaging performance of confocal microscope architectures via Monte Carlo simulations OPTICS LETTERS Chen, Y., Wang, D., Liu, J. T. C. 2012; 37 (21): 4495-4497

  Abstract

  Various confocal microscope architectures have been developed for in vivo tissue imaging, including single-axis confocal (SAC) and dual-axis confocal (DAC) configurations utilizing both point-scanning (PS) and line-scanning (LS) approaches. While it is known that these design variations lead to tradeoffs in imaging performance, a quantitative comparison of the imaging performance of these configurations in highly turbid media would be of value. Here, we perform Monte Carlo simulations to evaluate the optical-sectioning capability of these various confocal microscope architectures in reflectance mode. In particular, we investigate the axial and transverse responses of these configurations to reflective targets at various depths within a homogenous scattering medium. We find that the DAC-PS configuration results in superior rejection of multiply scattered background light compared to all other configurations, followed in performance by the SAC-PS, the DAC-LS, and then the SAC-LS. Line scanning with both the DAC and SAC configurations leads to photon crosstalk between pixels. However, at shallow depths, the axial and transverse resolution of all configurations is maintained in a homogeneous scattering medium.

  View details for DOI 10.1364/OL.37.004495

  View details for Web of Science ID 000310577700048

  View details for PubMedID 23114341

  View details for PubMedCentralID PMC3756678

• Identification of Cell Surface Targets through Meta-analysis of Microarray Data NEOPLASIA Haeberle, H., Dudley, J. T., Liu, J. T., Butte, A. J., Contag, C. H. 2012; 14 (7): 666-669

  Abstract

  High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

  View details for DOI 10.1593/neo.12634

  View details for Web of Science ID 000308489500010

  View details for PubMedID 22904683

  View details for PubMedCentralID PMC3421962

• Multi-color miniature dual-axis confocal microscope for point-of-care pathology OPTICS LETTERS Leigh, S. Y., Liu, J. T. C. 2012; 37 (12): 2430-2432

  Abstract

  We present a miniature microelectromechanical systems-based dual-axis confocal microscope capable of spatially coregistered fluorescence and reflectance imaging at multiple wavelengths. This device has a 10 mm diameter scan head with a 2 mm diameter tip for convenient use during surgery to guide tumor resection. The microscope has an adjustable focal depth of 20-200 micrometers and is capable of imaging with an axial resolution of 9 micrometers and in-plane resolution of 4 micrometers over a field of view of 450×450 micrometers. Simultaneous two-color imaging of individual optical sections is achieved by using a pair of grating-prism assemblies to compensate for chromatic dispersion in the 2 mm diameter gradient index relay lens at the distal tip of the device. Experimental measurements of the axial response of the microscope, as well as two-color images of a reflective bar target and fresh mouse brain tissues, demonstrate the performance of our device and its potential for multicolor in vivo optical sectioning microscopy.

  View details for DOI 10.1364/OL.37.002430

  View details for Web of Science ID 000305410500087

  View details for PubMedID 22739931

  View details for PubMedCentralID PMC3804107

• In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract JOURNAL OF BIOMEDICAL OPTICS Piyawattanametha, W., Ra, H., Qiu, Z., Friedland, S., Liu, J. T., Loewke, K., Kino, G. S., Solgaard, O., Wang, T. D., Mandella, M. J., Contag, C. H. 2012; 17 (2)

  Abstract

  Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm(2) and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

  View details for DOI 10.1117/1.JBO.17.2.021102

  View details for Web of Science ID 000303033600004

  View details for PubMedID 22463020

  View details for PubMedCentralID PMC3380818

• Point-of-care pathology with miniature microscopes ANALYTICAL CELLULAR PATHOLOGY Liu, J. T., Loewke, N. O., Mandella, M. J., Levenson, R. M., Crawford, J. M., Contag, C. H. 2011; 34 (3): 81-98

  Abstract

  Advances in optical designs are enabling the development of miniature microscopes that can examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to major changes in how diseases are detected and managed, driving a shift from today's diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to non-invasive point-of-care diagnosis with possible same-session definitive treatment. This shift may have major implications for the training requirements of future physicians to enable them to interpret real-time in vivo microscopic data, and will also shape the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires bridging gaps between clinicians, engineers and scientists. This article provides a forward-looking discussion of these issues, with a focus on malignant and pre-malignant lesions, by first highlighting some of the clinical areas where point-of-care in vivo microscopy could address unmet needs, and then by reviewing the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become a standard clinical tool.

  View details for DOI 10.3233/ACP-2011-0011

  View details for Web of Science ID 000293107200001

  View details for PubMedID 21673433

  View details for PubMedCentralID PMC3166958

• Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Loewke, N. O., Haeberle, H., Ra, H., Piyawattanametha, W., Solgaard, O., Kino, G. S., Contag, C. H. 2010; 15 (2)

  Abstract

  A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

  View details for DOI 10.1117/1.3386055

  View details for Web of Science ID 000278465300060

  View details for PubMedID 20459274

  View details for PubMedCentralID PMC2869369

• Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy BIOPHYSICAL JOURNAL Liu, J. T., Helms, M. W., Mandella, M. J., Crawford, J. M., Kino, G. S., Contag, C. H. 2009; 96 (6): 2405-2414

  Abstract

  The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.

  View details for DOI 10.1016/j.bpj.2008.12.3908

  View details for Web of Science ID 000266376700035

  View details for PubMedID 19289065

  View details for PubMedCentralID PMC2907718

• Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Crawford, J. M., Contag, C. H., Wang, T. D., Kino, G. S. 2008; 13 (3)

  Abstract

  Miniature endoscopic microscopes, with subcellular imaging capabilities, will enable in vivo detection of molecularly-targeted fluorescent probes for early disease detection. To optimize a dual-axis confocal microscope (DACM) design for this purpose, we use a tabletop instrument to determine the ability of this technology to perform optical sectioning deep within tissue. First, we determine how tissue scattering deteriorates the diffraction-limited transverse and vertical responses in reflectance imaging. Specifically, the vertical response of a DACM to a plane reflector is measured at various depths in a scattering phantom and compared with diffraction theory and Monte Carlo scattering simulations. Similarly, transverse line scans across a knife-edge target are performed at various depths in a scattering phantom. Second, as a practical demonstration of deep-tissue fluorescence microscopy that corroborates the findings from our scattering experiments, 3-D fluorescence images are obtained in thick human gastrointestinal mucosal specimens. Our results demonstrate efficient rejection of scattered light in a DACM, which enables deep optical sectioning in tissue with subcellular resolution that can distinguish between normal and premalignant pathologies.

  View details for DOI 10.1117/1.2939428

  View details for Web of Science ID 000257951200048

  View details for PubMedID 18601565

• Functional imaging of colonic mucosa with a fibered Confocal microscope for real-time in vivo pathology CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Wang, T. D., Friedland, S., Sahbaie, P., Soetikno, R., Hsiung, P., Liu, J. T., Crawford, J. M., Contag, C. H. 2007; 5 (11): 1300-1305

  Abstract

  Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time.Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified.Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09 microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 +/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001).Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation.

  View details for DOI 10.1016/j.cgh.2007.07.013

  View details for Web of Science ID 000250944900012

  View details for PubMedID 17936692

  View details for PubMedCentralID PMC2104519

• Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner OPTICS LETTERS Liu, J. T., Mandella, M. J., Ra, H., Wong, L. K., Solgaard, O., Kino, G. S., Piyawattanametha, W., Contag, C. H., Wang, T. D. 2007; 32 (3): 256-258

  Abstract

  The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

  View details for Web of Science ID 000244278900018

  View details for PubMedID 17215937

• Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia JOURNAL OF BIOMEDICAL OPTICS Liu, J. T., Mandella, M. J., Friedland, S., Soetikno, R., Crawford, J. M., Contag, C. H., Kino, G. S., Wang, T. D. 2006; 11 (5)

  Abstract

  A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-microm resolution, at tissue depths up to 610 microm. Post-objective scanning enables a large field of view (610 x 640 microm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.

  View details for DOI 10.1117/1.2363363

  View details for Web of Science ID 000242576900023

  View details for PubMedID 17092168

  View details for PubMedCentralID PMC2104521

• Near-infrared diode laser absorption diagnostic for temperature and water vapor in a scramjet combustor APPLIED OPTICS Liu, J. T., Rieker, G. B., Jeffries, J. B., Gruber, M. R., Carter, C. D., Mathur, T., Hanson, R. K. 2005; 44 (31): 6701-6711

  Abstract

  Tunable diode laser absorption measurements of gas temperature and water concentration were made at the exit of a model scramjet combustor fueled on JP-7. Multiplexed, fiber-coupled, near-infrared distributed feedback lasers were used to probe three water vapor absorption features in the 1.34-1.47 microm spectral region (2v1 and vl + v3 overtone bands). Ratio thermometry was performed using direct-absorption wavelength scans of isolated features at a 4-kHz repetition rate, as well as 2f wavelength modulation scans at a 2-kHz scan rate. Large signal-to-noise ratios demonstrate the ability of the optimally engineered optical hardware to reject beam steering and vibration noise. Successful measurements were made at full combustion conditions for a variety of fuel/air equivalence ratios and at eight vertical positions in the duct to investigate spatial uniformity. The use of three water vapor absorption features allowed for preliminary estimates of temperature distributions along the line of sight. The improved signal quality afforded by 2f measurements, in the case of weak absorption, demonstrates the utility of a scanned wavelength modulation strategy in such situations.

  View details for Web of Science ID 000232938000020

  View details for PubMedID 16270559

• Large-modulation-depth 2f spectroscopy with diode lasers for rapid temperature and species measurements in gases with blended and broadened spectra APPLIED OPTICS Liu, J. T., Jeffries, J. B., Hanson, R. K. 2004; 43 (35): 6500-6509

  Abstract

  A method that uses tunable diode lasers is developed for rapid temperature and concentration measurements of gases with highly broadened and congested spectra. Wavelength modulation absorption spectroscopy with 2f detection is utilized, because this derivative method offers benefits in dealing with blended spectral features. The 2f signal depends critically on the modulation depth of the laser alpha, which is increased to values above those typically achieved when wavelength modulation spectroscopy with diode lasers is performed. The 2f method with large modulation depths is validated by using near-IR diode lasers to probe pressure-broadened water-vapor features in the 1.4-microm region over a range of temperatures from 296 to 800 K and at pressures as high as 20 atm. Modulation depths as high as alpha = 0.8 cm(-1) are attained at modulation frequencies of 50 kHz and measurement bandwidths of 15 kHz. Comparisons of experimental results with 2f simulations, based on the HITRAN spectral database, provide confirmation of the capability of this method for rapid measurements of gas temperature and species concentration.

  View details for Web of Science ID 000225661400018

  View details for PubMedID 15617289