Fast 2-photon stimulation using holographic patterns

Session

Poster Session

Authors

Maia Brunstein (1), Jules Lubetzki (1), Cedric Moutoussamy (1), Wei Li (1), Jérémie Barral (1, 2)

Affiliations

1. Institut Pasteur, Université Paris Cité, INSERM, Institut de l’Audition, F-75012 Paris, France
2. CNRS

Keywords

Two-Photon imaging, Photostimulation, SLM, DMD, 3D Patterns

Abstract text

A fundamental question in neuroscience is to understand how patterns of neuronal activity can represent useful sensory or motor information to drive animal behavior. To build a causal link between brain activity and behavior, it is necessary to control the neuronal dynamics and observe how animal comportment is affected [1]. Addressing this challenge requires tools to manipulate a population of neurons in a very controlled manner, both spatially and temporally. This can now be achieved by optogenetics, a genetic manipulation that drives the expression of a light-modulated actuator in a defined cell type in order to modify cellular properties during illumination. A variety of approaches have been developed to generate precise spatiotemporal light patterns to stimulate the neurons on the brain. Yet certain constrains still exists in the current optical techniques to activate a neuronal population with both cellular resolution and millisecond precision. Here, we describe an experimental setup allowing to stimulate a few tens of neurons in a plane at sub-millisecond rates using 2-photon activation. We used a LCoS-SLM to generate a single spatial pattern in 2 or 3 dimensions containing multiple regions of interest and projected this pattern onto a DMD that was employed as a fast temporal shutter for each region of interest. In this way, the total laser power was efficiently dedicated to activating only potential targets without wasting laser power on unused DMD pixels. This approach had been applied to imaging application with the development of an encoded multisite 2P microscope [2] or to multi-site photolysis of caged neurotransmitters [3]. The main advantages of our optical developments are (i) to push the temporal resolution to the limitations of the opsin time constant, (ii) to reduce the complexity of the phase pattern calculation since only 1 phase pattern needs to be built, (iii) to enable real-time control of neuronal activity. 

Using fluorescent microscopy and patch-clamp recording of neurons in culture expressing the light-gated ion channels, we characterized the temporal and spatial resolution of the microscope. We described the advantages of combining the LCoS-SLM with the DMD to maximize the temporal precision, modulate the illumination amplitude, and reduce background activation. We further demonstrate that this approach can be extended to patterns in 3 dimensions (see figure) [4]. This methodology is well suited to address important questions about the role of temporal information in neuronal coding.

Figure: Selective modulation of patterns in 3 dimensions. (a) 3-dimensional reconstruction (top) of the optical patterns when all the mirrors of the DMD where in the ON position. Two selected planes are shown (bottom). (b-d) Same as in (a) when only the mirrors corresponding to the 8 ROIs where ON (b) or when only 7 (c) or 6 (d) ROIs were ON on the DMD.

References

1. S. Panzeri, C. D. Harvey, E. Piasini, P. E. Latham, and T. Fellin, “Cracking the Neural Code for Sensory Perception by Combining Statistics, Intervention, and Behavior,” Neuron 93(3), 491–507 (2017)

2. M. Ducros, Y. Goulam Houssen, J. Bradley, V. de Sars, and S. Charpak, “Encoded multisite two-photon microscopy,” Proc. Natl. Acad. Sci. U. S. A. 110(32), 13138–13143 (2013).

3. M. A. Go, M. S. To, C. Stricker, S. Redman, H. A. Bachor, G. J. Stuart, and V. R. Daria, “Four-dimensional multi-site photolysis of caged neurotransmitters,” Front. Cell. Neurosci. 7, 231 (2013).

4. "Fast 2-photon stimulation using holographic patterns", Maia Brunstein, Jules Lubetzki, Cédric Moutoussamy, Wei Li, and Jérémie Barral, Optics Express, Vol. 31, Issue 23, pp. 39222-39238 (2023), https://doi.org/10.1364/OE.498644