Multiplexed temporally focused light shaping for high-resolution multi-cell targeting

Abstract

15Patterning light at the single-cell level over multiple neurons in the brain is crucial for optogenetic photostimulation that can recapitulate natural activity patterns and, thereby, determine the role of specific components of brain activity in behavior. To this end we have developed a method for projecting three-dimensional, 2-photon excitation patterns that are confined to many individual neurons. The new versatile optical scheme generates multiple extended excitation spots in a large volume with micrometric 20 lateral and axial resolution. Two-dimensional temporally focused shapes are multiplexed several times over selected positions, thanks to the precise spatial phase modulation of the pulsed beam. This permits, under multiple configurations, the generation of tens of axially confined spots in an extended volume, spanning a range in depth of up to 500 µm. We demonstrate the potential of the approach by performing multi-cell volumetric excitation of photoactivatable GCaMP in the central nervous system of Drosophila 25 larvae, a challenging structure with densely arrayed and small diameter neurons, and by photoconverting the fluorescent protein Kaede in zebrafish larvae. Our technique paves the way for the optogenetic manipulation of a large number of neurons in intact circuits.

Multiplexed temporally focused light shaping through a GRIN lens for precise in-depth optogenetic photostimulation

“…the x direction) when moving away from the centre much faster than for the y direction. This asymmetry, as detailed in the discussion, was not observed in previous experiments 27 , and could be due to a loss of colours through the GRIN lens when displacing the holographic spot in the TF direction. In any case, the temporally focused spots maintained an axial resolution better than 25 µm across 120 µm in the y direction and across 60 µm in the x direction, reaching the value of 35 µm at 60 µm from the centre along x. spots are focused at different depths.…”

“…It had a diameter of 0.5 mm and a total length of 9.86 mm, with NA of 0.19 on the objective side and of 0.5 on the sample side, resulting in a total field of view (FOV) of As discussed in Ref. 27 , the MTF-LS technique is compatible with several different beam-shaping strategies that one can perform with the first SLM. In the following we show the results of (i) multiplexed temporally focused computer generated holography (MTF-CGH) and (ii) multiplexed temporally focused multi shapes (MTF-MS) through the GRIN lens.…”

“…As described in Ref. 27 , the MTF-MS method relies on a mixed phase/amplitude shaping approach, capable of simultaneously producing different speckle-free shapes, with an overall better axial resolution with respect to MTF-CGH, and to independently multiplex them at the sample plane. Here we used MTF-MS in two different configurations ( Fig.…”

“…This was done either by generating multiple diffraction-limited spots that were scanned simultaneously across multiple cell somata [18][19][20] , or by using computer generate holography (CGH) to produce light patterns covering multiple cell somata at once 21 , thus optimizing the temporal precision of the photostimulation 22 . Recently, several research groups [23][24][25][26][27] have shown that using a two-step wave front shaping combined with temporal focusing (TF) [28][29][30][31] it is possible to generate multiple high resolution extended light patterns in 3D, a technique we named multiplexed temporally focused light shaping (MTF-LS). These approaches led to the first demonstrations of neural circuit manipulation in 3D 25,26 , yet the need of using conventional high numerical aperture (NA) objectives limited their use to circuits in superficial (≤ 300 µm) cortical areas or to in-vitro applications.…”

“…In this work we demonstrate an optical system combining our recent MTF-LS technique 27 with the use of a GRIN lens (NA=0.5, diameter 0.5 mm), and we show that a small ME is suitable for the generation of multiple axially-confined 2P excitation spots matching target somata in 3D. Successively, using this system we demonstrate all-optical control of single or multiple neurons through the ME, by performing in vivo 2P optogenetic stimulation of target cells and reading out their induced activity with 2P calcium imaging.…”