Honors & Awards


  • Best Poster Presentation, International Chemical Biology Society, Madison, Wisconsin (2016)
  • National Science Scholarship (PhD), A*STAR, Singapore (2014-2019)
  • Edna H Graham’41 Prize, Mount Holyoke College (2013)
  • Phi Beta Kappa, Mount Holyoke College (2013)
  • Roll of Honors, A*STAR, Singapore (2013)
  • Abby Howe Turner Award for Excellence in Biological Sciences, Mount Holyoke College (2012)
  • Louisa Stone Stephenson Prizes for Excellence in Chemistry, Mount Holyoke College (2012)
  • Chairman's Honors List, A*STAR, Singapore (2011-2013)
  • Bernice MacLean Award for Excellence in Biological Sciences, Mount Holyoke College (2011-2012)
  • National Science Scholarship (BS), A*STAR, Singapore (2010-2013)

Professional Education


  • Doctor of Philosophy, University of California Berkeley (2019)
  • BA, Mount Holyoke College, Biochemistry (2013)
  • PhD, University of California, Berkeley, Chemistry (2019)

Stanford Advisors


All Publications


  • Electrophysiology, Unplugged: Imaging Membrane Potential with Fluorescent Indicators. Accounts of chemical research Liu, P., Miller, E. W. 2020; 53 (1): 11–19

    Abstract

    Membrane potential is a fundamental biophysical property maintained by every cell on earth. In specialized cells like neurons, rapid changes in membrane potential drive the release of chemical neurotransmitters. Coordinated, rapid changes in neuronal membrane potential across large numbers of interconnected neurons form the basis for all of human cognition, sensory perception, and memory. Despite the importance of this highly orchestrated and distributed activity, the traditional method for recording membrane potential is through the use of highly invasive single-cell electrodes that offer only a small glimpse of the total activity within a system. Fluorescent dyes that change their optical properties in response to changes in biological voltage have the potential to provide a powerful complement to traditional electrode-based methods of inquiry. Voltage-sensitive fluorescent indicators would allow the direct observation of membrane potential changes, significantly expanding our ability to monitor membrane potential dynamics in living systems. Toward this end, we have initiated a program to design, synthesize, and apply voltage-sensitive fluorophores that report on membrane potential dynamics with high sensitivity and speed. The basis for this optical voltage sensing is membrane potential-dependent photoinduced electron transfer (PeT). Voltage-sensitive fluorophores, or VoltageFluors, possess a fluorophore, a conjugated molecular wire, and an aniline donor. At resting potentials, in which the cell has a hyperpolarized or negative potential relative to the outside of the cell, PeT from the aniline donor is enhanced and fluorescence is diminished. At depolarized potentials, the membrane potential decreases the rate of PeT, allowing an increase in fluorescence. We show that a number of different fluorophores, molecular wires, and aniline donors can be employed to generate fast and sensitive VoltageFluor dyes. Multiple lines of evidence point to a PeT-based mechanism for voltage sensing, delivering fast response kinetics (∼25 ns), good sensitivity (>60% ΔF/F), compatibility with two-photon illumination, excellent signal-to-noise, and the ability to detect neuronal and cardiac action potentials in single trials. In this Account, we provide an overview of the challenges facing the design of fluorescent voltage indicators. We trace the development of molecular wire-based fluorescent voltage indicators within our group, beginning from fluorescein-based VoltageFluor to long-wavelength indicators that use modern fluorophores like silicon rhodamine and carbofluorescein. We examine design principles for PeT-based voltage indicators, showcase the use of our recent indicators for two-photon voltage imaging in intact brains, and explore the development of hybrid indicators that can localize to genetically defined cells. Finally, we highlight outstanding challenges to and opportunities for voltage imaging.

    View details for DOI 10.1021/acs.accounts.9b00514

    View details for PubMedID 31834772

  • Water-Soluble BODIPY Photocages with Tunable Cellular Localization. Journal of the American Chemical Society Kand, D., Liu, P., Navarro, M. X., Fischer, L. J., Rousso-Noori, L., Friedmann-Morvinski, D., Winter, A. H., Miller, E. W., Weinstain, R. 2020; 142 (11): 4970–74

    Abstract

    Photoactivation of bioactive molecules allows manipulation of cellular processes with high spatiotemporal precision. The recent emergence of visible-light excitable photoprotecting groups has the potential to further expand the established utility of the photoactivation strategy in biological applications by offering higher tissue penetration, diminished phototoxicity, and compatibility with other light-dependent techniques. Nevertheless, a critical barrier to such applications remains the significant hydrophobicity of most visible-light excitable photocaging groups. Here, we find that applying the conventional 2,6-sulfonation to meso-methyl BODIPY photocages is incompatible with their photoreaction due to an increase in the excited state barrier for photorelease. We present a simple, remote sulfonation solution to BODIPY photocages that imparts water solubility and provides control over cellular permeability while retaining their favorable spectroscopic and photoreaction properties. Peripherally disulfonated BODIPY photocages are cell impermeable, making them useful for modulation of cell-surface receptors, while monosulfonated BODIPY retains the ability to cross the cellular membrane and can modulate intracellular targets. This new approach is generalizable for controlling BODIPY localization and was validated by sensitization of mammalian cells and neurons by visible-light photoactivation of signaling molecules.

    View details for DOI 10.1021/jacs.9b13219

    View details for PubMedID 32115942

  • Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators. Journal of the American Chemical Society Grenier, V., Daws, B. R., Liu, P., Miller, E. W. 2019; 141 (3): 1349–58

    Abstract

    Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.

    View details for DOI 10.1021/jacs.8b11997

    View details for PubMedID 30628785

    View details for PubMedCentralID PMC6475477

  • Covalently tethered rhodamine voltage reporters for high speed functional imaging in brain tissue. Journal of the American Chemical Society Deal, P. E., Liu, P., Al-Abdullatif, S. H., Muller, V. R., Shamardani, K., Adesnik, H., Miller, E. W. 2019

    Abstract

    Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemical synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically-encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer (PeT) triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethyleneglycol (PEG) linker (RhoVR-Halos). When applied to cells, RhoVR-Halos selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halos maintain high voltage sensitivities-up to 34% ΔF/F per 100 mV-and fast response times typical of untargeted RhoVRs, while gaining the selectivity typical of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca2+ indicators like GCaMP to provide simultaneous voltage and Ca2+ imaging. In brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read out of action potentials from labeled cortical neurons in rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically-encodable systems, permitting imaging modalities inaccessible to either technique individually.

    View details for DOI 10.1021/jacs.9b12265

    View details for PubMedID 31829585

  • Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging. Journal of the American Chemical Society Ortiz, G., Liu, P., Naing, S. H., Muller, V. R., Miller, E. W. 2019; 141 (16): 6631–38

    Abstract

    We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p Ka of carbofluorescein dyes, coupled with their tendency to cyclize to nonfluorescent configurations, precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p Ka by 2 units to 5.2 and sulfonation to prevent cyclization to the nonabsorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p Ka values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Δ F/ F per 100 mV), and can be used in the presence of other blue-excited fluorophores such as green fluorescent protein. Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.

    View details for DOI 10.1021/jacs.9b01261

    View details for PubMedID 30978010

    View details for PubMedCentralID PMC6546115

  • Directing GDNF-mediated neuronal signaling with proactively programmable cell-surface saccharide-free glycosaminoglycan mimetics. Chemical communications (Cambridge, England) Cai, S., Lukamto, D. H., Toh, J. K., Huber, R. G., Bond, P. J., Jee, J. E., Lim, T. C., Liu, P., Chen, L., Qu, Q. V., Lee, S. S., Lee, S. G. 2019; 55 (9): 1259–62

    Abstract

    A significant barrier to harnessing the power of cell-surface glycosaminoglycans (GAGs) to modulate glial cell-line-derived neurotrophic factor (GDNF) signaling is the difficulty in accessing key GAG structures involved. Here, we report tailored GDNF signaling using synthetic polyproline-based GAG mimetics (PGMs). PGMs deliver the much needed proactive programmability for GDNF recognition and effectively modulate GDNF-mediated neuronal processes in a cellular context.

    View details for DOI 10.1039/c8cc09253b

    View details for PubMedID 30632548

  • Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons. Journal of the American Chemical Society Liu, P., Grenier, V., Hong, W., Muller, V. R., Miller, E. W. 2017; 139 (48): 17334–40

    Abstract

    We present a method to target voltage-sensitive fluorescent dyes to specified cells using an enzyme-catalyzed fluorogenic reaction on cell surfaces. The dye/enzyme hybrids are composed of a photoinduced electron transfer (PeT)-based fluorescent voltage indicator and a complementary enzyme expressed on the cell surface. Action of the exogenous enzyme on the dye results in fluorogenic activation of the dye, enabling fast voltage imaging in defined neurons with sensitivity surpassing those of purely genetically encoded approaches. We employ a bulky methylcyclopropylacetoxymethyl ether to diminish the fluorescence of a PeT-based voltage-sensitive dye, or VoltageFluor. The hydrolytically stable ether can be removed by the action of porcine liver esterase (PLE) to reveal the bright unmodified VoltageFluor. We established that the chemically modified VoltageFluor is a substrate for PLE in vitro and in live cells. When PLE is targeted to the external face of cell membranes, it controls the apparent staining of cells. The use of neuron-specific promoters can direct staining to mammalian neurons to provide clear detection of neuronal action potentials in single trials. All of the new VoltageFluors targeted by esterase expression (VF-EXs) report single spikes in cultured mammalian neurons. The best, VF-EX2, does so with a signal-to-noise ratio nearly double that of comparable genetically encoded voltage reporters. By targeting PLE to neurons, VF-EX2 can interrogate the neuromodulatory effects of serotonin in cultured hippocampal neurons. Taken together, our results show that a combination of synthetic chemistry and biochemistry enables bright and fast voltage imaging from genetically defined neurons in culture.

    View details for DOI 10.1021/jacs.7b07047

    View details for PubMedID 29154543

    View details for PubMedCentralID PMC5718920

  • Tailored chondroitin sulfate glycomimetics via a tunable multivalent scaffold for potentiating NGF/TrkA-induced neurogenesis. Chemical science Liu, P., Chen, L., Toh, J. K., Ang, Y. L., Jee, J. E., Lim, J., Lee, S. S., Lee, S. G. 2015; 6 (1): 450–56

    Abstract

    The challenges inherent in the synthesis of large glycosaminoglycan (GAG) polysaccharides have made chemically accessible multivalent glycoligands a valuable tool in the field of GAG mimetics. However, the difficulty of positioning sulfated sugar motifs at desired sites has hindered efforts to precisely tailor their biofunctions. Here, we achieved precise orientation of sulfated disaccharide motifs by taking advantage of a structurally well-defined polyproline scaffold, and describe systematic explorations into the importance of the spatial arrangement of sulfated sugars along the scaffold backbone in designing multivalent glycoligands. Our protein binding studies demonstrate that the specific conformational display of pendant sugars is central to direct their multivalent interactions with NGF. By employing computational modeling and cellular studies, we have further applied this approach to engineer NGF-mediated signaling by regulating the NGF/TrkA complexation process, leading to enhanced neuronal differentiation and neurite outgrowth of PC12 cells. Our findings offer a promising strategy for the pinpoint engineering of GAG-mediated biological processes and a novel method of designing new therapeutic agents that are highly specific to GAG-associated disease.

    View details for DOI 10.1039/c4sc02553a

    View details for PubMedID 28694940

    View details for PubMedCentralID PMC5485393

  • meso-Methylhydroxy BODIPY: a scaffold for photo-labile protecting groups. Chemical communications (Cambridge, England) Rubinstein, N., Liu, P., Miller, E. W., Weinstain, R. 2015; 51 (29): 6369–72

    Abstract

    Here, we show that by installing a meso-methylhydroxy moiety, the boron dipyrromethene (BODIPY) scaffold can be converted into an efficient caging group, removable by green light. We describe caging and uncaging of important chemical functionalities and demonstrate green light mediated control over biological processes in cultured cell lines and neurons.

    View details for DOI 10.1039/c5cc00550g

    View details for PubMedID 25761909