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Elric Esposito, Giulia Spampinato, Pierre Yger, Deniz Dalkara, Emiliano Ronzitti, Eirini Papagiakoumou, Valentina Emiliani, Serge A Picaud, Olivier Marre, Jens Duebel; Advanced Neural Circuit Analysis by Combining High-Resolution Optogenetic Stimulation, Two-Photon Imaging and Electrophysiology. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2586.
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© ARVO (1962-2015); The Authors (2016-present)
Investigation of retinal network activity of various retinal cells types remains a challenge, given current tools available. We developed a versatile optical instrument that allows for patterned single-photon (1P) / two-photon (2P) stimulation and simultaneous monitoring of neural activity through 2P imaging and electrophysiological recording.
We combined a two-photon (2P) laser scanning imaging system with a two-photon stimulation scheme using Spatial Light Modulator (SLM) based digital holography. The vast tuning range of the first laser output (680-1300nm) allows 2P imaging of a wide range of fluorophores, where the second output (fixed 1040 nm) is used for 2P stimulation of specific cells, transfected with optogenetic tools. The system further benefits from a digital micromirror device (DMD) projecting complex light pattern through the same high numerical aperture (NA = 1) objective. For electrophysiology, the setup is equipped with a patch-clamp rig for single cell analysis and a multi-electrode array (MEA) for population spike recording.
The system provided high resolution 2P imaging (~0.5 mm XY, <2 mm Z) and 2P stimulation with an axial confinement under 50 μm for a 10 μm diameter spot. The setup was characterized through the MEA by patch-clamp recording of Human Embryonic Kidney (HEK) cells, transfected with a red-shifted microbial opsin (ReaChR) and stimulated at 1040 nm. The system was also used to stimulate retinal interneuron cell activity while recording a large population of RGC’s using the MEA.
We have designed a custom made optical system that not only can observe several retinal layers at high resolution, but can also control the activity of cells of interest, while recording electrical activity of post-synaptic cells. Combined with various optogenetic tools that can be expressed in specific cell types, our method will help to investigate the functional connections of neural microcircuits.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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