Parallelized computational 3D video microscopy of freely moving organisms at multiple gigapixels per second

To study the behavior of freely moving model organisms such as zebrafish (Danio rerio) and fruit flies (Drosophila) across multiple spatial scales, it would be ideal to use a light microscope that can resolve 3D information over a wide field of view (FOV) at high speed and high spatial resolution. However, it is challenging to design an optical instrument to achieve all of these properties simultaneously. Existing techniques for large-FOV microscopic imaging and for 3D image measurement typically require many sequential image snapshots, thus compromising speed and throughput. Here, we present 3D-RAPID, a computational microscope based on a synchronized array of 54 cameras that can capture high-speed 3D topographic videos over a 135-cm^2 area, achieving up to 230 frames per second at throughputs exceeding 5 gigapixels (GPs) per second. 3D-RAPID features a 3D reconstruction algorithm that, for each synchronized temporal snapshot, simultaneously fuses all 54 images seamlessly into a globally-consistent composite that includes a coregistered 3D height map. The self-supervised 3D reconstruction algorithm itself trains a spatiotemporally-compressed convolutional neural network (CNN) that maps raw photometric images to 3D topography, using stereo overlap redundancy and ray-propagation physics as the only supervision mechanism. As a result, our end-to-end 3D reconstruction algorithm is robust to generalization errors and scales to arbitrarily long videos from arbitrarily sized camera arrays. The scalable hardware and software design of 3D-RAPID addresses a longstanding problem in the field of behavioral imaging, enabling parallelized 3D observation of large collections of freely moving organisms at high spatiotemporal throughputs, which we demonstrate in ants (Pogonomyrmex barbatus), fruit flies, and zebrafish larvae

Modulation du front d’onde pour l’excitation biphotonique en optogénétique

International audienceLe développement de l'optogénétique en neurosciences L'expression ciblée de protéines photo-sensibles sous contrôle d'un promoteur spécifique, technique que l'on désigne sous l'appellation optogénétique, a connu un essor formidable ces dernières années dans le domaine des neuroscien-ces. La diversité des protéines photo-sensibles naturelles et leur amélioration par ingénierie génétique ont permis de créer une vaste gamme d'outils opto-génétiques qui permettent de contrôler l'activité cérébrale avec la lumière, via la stimulation ou l'inhibition de neurones. L'optogénétique s'est imposée et a pris peu à peu l'avantage sur des méthodes traditionnelles fondées sur la pose d'électrodes et la pharmacologie, en exploitant les avantages des techniques de stimulation optique. En effet, contrairement aux électrodes, la photostimulation ne provoque pas de perturbations mécaniques et permet d'activer plusieurs régions avec une plus grande flexibilité et une meilleure précision spatiale. L'outil optogénétique le plus couramment utilisé est un canal cationique excitateur qui s'insère dans la membrane plasmique, la channelrhodopsin-2 (ChR2). Grâce au faible niveau d'intensité lumineuse requis pour son excitation (1 mW/mm², lumière bleue), la ChR2 a pu être activée avec une illumination à champ large délivrée, par exemple, par des fibres optiques implantées dans le cerveau d'animaux in vivo. La ChR2 a ainsi grandement contribué à notre compréhension des mécanismes responsables de pathologies complexes comme l'anxiété [1], et a permis de relier l'activité de certains types neuronaux à des comportements précis, comme dans le cas des neurones cholinergiques du noyau accumbens [2]. Pourtant une illumination à champ large n'est pas suffisante pour répondre à certaines questions, concernant par exemple la connectivité interne des circuits neuronaux, qui demande une résolution cellulaire ou subcellulaire. Un contrôle tridimensionnel de l'illumination permettrait de sélectionner un sous-groupe de neurones parmi ceux qui expriment le promoteur lié aux protéines photosensibles, ou même de cibler un compartiment cellulaire comme une dendrite. Cette précision est nécessaire pour analyser l'intégration des signaux des différents compartiments cellulaires et comprendre le traitement de l'information par le neurone

Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation

International audienceThe use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1

Functional patterned multiphoton excitation deep inside scattering tissue

International audienceStochastic distortion of light beams in scattering samples makes in-depth photoexcitation in brain tissue a major challenge. A common solution for overcoming scattering involves adaptive pre-compensation of the unknown distortion1,2,3. However, this requires long iterative searches for sample-specific optimized corrections, which is a problem when applied to optical neurostimulation where typical timescales in the system are in the millisecond range. Thus, photoexcitation in scattering media that is independent of the properties of a specific sample would be an ideal solution. Here, we show that temporally focused two-photon excitation4 with generalized phase contrast5 enables photoexcitation of arbitrary spatial patterns within turbid tissues with remarkable robustness to scattering. We demonstrate three-dimensional confinement of tailored photoexcitation patterns >200 µm in depth, both in numerical simulations and through brain slices combined with patch-clamp recording of photoactivated channelrhodopsin-2

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species

Scanless two-photon excitation of channelrhodopsin-2

International audienceLight-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (tF-GPc) to shape two-photon excitation for this purpose. the illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. the tF-GPc two-photon excitation patterns generated large photocurrents in channelrhodopsin-2-expressing cultured cells and neurons and in mouse acute cortical slices. the amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials