PhD, Université Paris Descartes - Université Sorbonne Paris Cité, Physics (2016)
Master of Biomedical Engineering, Télécom ParisTech, Bioimaging (2012)
Telecommunications Engineering, Universidad de Cantabria, Radio Communications (2011)
Mark Schnitzer, Postdoctoral Faculty Sponsor
Kilohertz two-photon brain imaging in awake mice.
Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.
View details for DOI 10.1038/s41592-019-0597-2
View details for PubMedID 31659327
Optogenetic stimulation of complex spatio-temporal activity patterns by acousto-optic light steering probes cerebellar granular layer integrative properties
2018; 8: 13768
Optogenetics provides tools to control afferent activity in brain microcircuits. However, this requires optical methods that can evoke asynchronous and coordinated activity within neuronal ensembles in a spatio-temporally precise way. Here we describe a light patterning method, which combines MHz acousto-optic beam steering and adjustable low numerical aperture Gaussian beams, to achieve fast 2D targeting in scattering tissue. Using mossy fiber afferents to the cerebellar cortex as a testbed, we demonstrate single fiber optogenetic stimulation with micron-scale lateral resolution, >100 µm depth-penetration and 0.1 ms spiking precision. Protracted spatio-temporal patterns of light delivered by our illumination system evoked sustained asynchronous mossy fiber activity with excellent repeatability. Combining optical and electrical stimulations, we show that the cerebellar granular layer performs nonlinear integration, whereby sustained mossy fiber activity provides a permissive context for the transmission of salient inputs, enriching combinatorial views on mossy fiber pattern separation.
View details for PubMedID 30213968
Three-dimensional spatiotemporal focusing of holographic patterns
Two-photon excitation with temporally focused pulses can be combined with phase-modulation approaches, such as computer-generated holography and generalized phase contrast, to efficiently distribute light into two-dimensional, axially confined, user-defined shapes. Adding lens-phase modulations to 2D-phase holograms enables remote axial pattern displacement as well as simultaneous pattern generation in multiple distinct planes. However, the axial confinement linearly degrades with lateral shape area in previous reports where axially shifted holographic shapes were not temporally focused. Here we report an optical system using two spatial light modulators to independently control transverse- and axial-target light distribution. This approach enables simultaneous axial translation of single or multiple spatiotemporally focused patterns across the sample volume while achieving the axial confinement of temporal focusing. We use the system's capability to photoconvert tens of Kaede-expressing neurons with single-cell resolution in live zebrafish larvae.
View details for DOI 10.1038/ncomms11928
View details for Web of Science ID 000378814400001
View details for PubMedID 27306044
View details for PubMedCentralID PMC4912686
Zero-order suppression for two-photon holographic excitation
2014; 39 (20): 5953-5956
Wavefront shaping with liquid-crystal spatial light modulators (LC-SLMs) is frequently hindered by a remaining fraction of undiffracted light, the so-called "zero-order." This contribution is all the more detrimental in configurations for which the LC-SLM is Fourier conjugated to a sample by a lens, because in these cases this undiffracted light produces a diffraction-limited spot at the image focal plane. In this Letter we propose to minimize two-photon (2P) excitation of the sample, resulting from this unmodulated light, by introducing optical aberrations to the excitation beam. Aberrations are subsequently compensated by the LC-SLM, but only for the modulated part of the beam, and not for the zero-order component. In order to experimentally demonstrate the method, we use astigmatism as the optical aberration, by simply adding one or two cylindrical lenses in the optical path of the beam. A 10⁴ decrease in zero-order-induced 2P fluorescence intensity is demonstrated. Combining this approach with temporal focusing is shown to decrease zero-order fluorescence by a factor of 4·10⁶.
View details for DOI 10.1364/OL.39.005953
View details for Web of Science ID 000343912000043
View details for PubMedID 25361128
When can temporally focused excitation be axially shifted by dispersion?
2014; 22 (6): 7087-7098
Temporal focusing (TF) allows for axially confined wide-field multi-photon excitation at the temporal focal plane. For temporally focused Gaussian beams, it was shown both theoretically and experimentally that the temporal focus plane can be shifted by applying a quadratic spectral phase to the incident beam. However, the case for more complex wave-fronts is quite different. Here we study the temporal focus plane shift (TFS) for a broader class of excitation profiles, with particular emphasis on the case of temporally focused computer generated holography (CGH) which allows for generation of arbitrary, yet speckled, 2D patterns. We present an analytical, numerical and experimental study of this phenomenon. The TFS is found to depend mainly on the autocorrelation of the CGH pattern in the direction of the beam dispersion after the grating in the TF setup. This provides a pathway for 3D control of multi-photon excitation patterns.
View details for DOI 10.1364/OE.22.007087
View details for Web of Science ID 000333579300088
View details for PubMedID 24664057
Quantitative Imaging of Microtubule Alteration as an Early Marker of Axonal Degeneration after Ischemia in Neurons
2013; 104 (5): 968-975
Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.
View details for DOI 10.1016/j.bpj.2013.01.020
View details for Web of Science ID 000315748800005
View details for PubMedID 23473479
- Analysis of optical crosstalk in flexible imaging endoscopes based on multicore fibers Conference on Biophotonics - Photonic Solutions for Better Health Care II SPIE-INT SOC OPTICAL ENGINEERING. 2010