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Article
Three-dimensional Multi-site Random Access Photostimulation (3D-MAP)
Hillel Adesnik
Berkeley
Corresponding Author
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Optical control of neural ensemble activity has been crucial for understanding brain function and disease, yet no technology can achieve optogenetic control of very large numbers of neurons at extremely fast rates over a large volume. State-of-the-art multiphoton holographic optogenetics requires high power illumination that only address relatively small populations of neurons in parallel. Conversely, one-photon holographic techniques can stimulate more neurons but with a trade-off between resolution and addressable volume. We introduce a new one-photon light sculpting technique, termed Three-Dimensional Multi-site random Access Photostimulation (3D-MAP), that simultaneously overcomes all these limitations by dynamically modulating light in both the spatial and angular domain at multi-kHz rates. Electrophysiological measurements confirm that 3D-MAP achieves high spatial precision in vitro and in vivo. Using 3D-MAP, we then interrogate neural circuits with 3D multi-site illumination with high resolution over a large volume of intact brain that existing techniques cannot achieve.
Keywords
3D-MAP, optogenetic, 3D multi-site illumination
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This is a list of supplementary files associated with this preprint. Click to download.
- ReportingSummary.pdf
Reporting Summary
- FigS1.png
Fig. S1. Comparison of 3D spatial resolution of 3D-MAP versus existing photostimulation approaches. All simulations are aim to generate a 10µm-diameter spot in-focus to match the size of a neuron in order to maximize photostimulation efficiency. The light fields are calculated with 635nm red light illumination and 20x, NA = 1.0 objective lens. The pixel size of the DMD is 7.5µm. The number of pixels of the SLM for CGH is 1152x1152 pixels. (a, b) The lateral cross-section (a) and axial cross-section (b) of CGH at NA = 1.0 (overfill the back aperture). The FOV under high NA illumination is 319x319µm2, which is much smaller than the FOV of 3D-MAP. (c, d) The lateral cross-section (c) and axial cross-section (d) of CGH at effective NA = 0.55 (under-fill the back aperture) to match the FOV of 3D-MAP (800x800µm2). (e, f) The lateral cross-section (e) and axial cross-section (f) of spiral scan with a single focus. The total intensity is an incoherent sum of the intensity of each scanning point. (g, h) The lateral cross-section (g) and axial cross-section (h) of 2D DMD projection. (i, j) The lateral cross-section (i) and axial cross-section (j) of 3D-MAP (10 overlapping beams). (k, l) A comparison of intensity profile of all these methods along the x-axis (k) and z-axis (l). 3D-MAP achieves the highest 3D spatial resolution among all the methods. Figure (a-j) are in the same size of 100x100µm2. Scale bar, 10µm.
- FigS2.png
Fig. S2. 3D-MAP is able to simultaneously generate multiple foci anywhere in 3D. (a) Five foci are located right on top of each other along the z-axis. Left: XZ cross-section. Right: XY cross-section corresponding to the z depths marked with green dash lines. (b) Nine foci with evenly distributed depths form a tilted plane across the 3D volume. For both (a) and (b), the target location is indicated in the 3D diagram on the left, and the corresponding experimental 3D fluorescence measurements are on the right. The foci are recorded in 3D by capturing a stack of 2D fluorescence images at various depths of the 3D illumination pattern intercepting a thin, uniform, fluorescent calibration slide with a sub-stage objective coupled to a camera. Scale bar, 100μm.
- FigS3.png
Fig. S3. 3D-MAP provides mapping of inhibitory synaptic connections in vivo. We patched a pyramidal neuron without opsin and photo-stimulated the volume around it pixel-by-pixel, and the parvalbumin neurons connect to this patched cell via synapses expressed opsin. The current readout reveals the inhibitory synaptic connections from all parvalbumin neurons to this pyramidal neuron. The mapping process is repeated 4 times. (a) Each row of the images is measured under the same stimulation laser power (100% power is 419μW), and each column of the images is at the same axial plane. Scale bar: 100µm. (b) Representative traces of postsynaptic currents under each stimulation power (measured from the pixel pointed by white arrow). These traces elucidate the synaptic connection is binary: once the neuron is photo-stimulated, the synaptic current remains the same, even if the stimulation power is increased. The unique photosensitivity of each neuron helps us to identify different neural ensembles by changing the stimulation power.
- FigS4.png
Fig. S4. Multi-site random simultaneous stimulation by 3D-MAP can reconstruct synaptic connectivity maps with fewer measurements than single-target stimulation. M: number of repeat measurements (see Computational reconstruction framework in Methods). The data is recorded from five simultaneously stimulated foci. The optimization problem is underdetermined for values of M < 5. Assuming the five foci stimulate sites which are sparsely distributed in space, it is possible to reconstruct the synaptic connectivity map with fewer measurements (M=1-4) using compressive sensing. Scale bar, 100μm.
- TableS1.png
Supplementary materials Table 1. Comparison of light-targeting photostimulation methods
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Article
Three-dimensional Multi-site Random Access Photostimulation (3D-MAP)
Hillel Adesnik
Berkeley
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 28 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Photonics/optics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Optical control of neural ensemble activity has been crucial for understanding brain function and disease, yet no technology can achieve optogenetic control of very large numbers of neurons at extremely fast rates over a large volume. State-of-the-art multiphoton holographic optogenetics requires high power illumination that only address relatively small populations of neurons in parallel. Conversely, one-photon holographic techniques can stimulate more neurons but with a trade-off between resolution and addressable volume. We introduce a new one-photon light sculpting technique, termed Three-Dimensional Multi-site random Access Photostimulation (3D-MAP), that simultaneously overcomes all these limitations by dynamically modulating light in both the spatial and angular domain at multi-kHz rates. Electrophysiological measurements confirm that 3D-MAP achieves high spatial precision in vitro and in vivo. Using 3D-MAP, we then interrogate neural circuits with 3D multi-site illumination with high resolution over a large volume of intact brain that existing techniques cannot achieve.
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