Two-photon imaging via implanted lenses was performed as previously described (Lutas et al., 2019 (link)) with the following adjustments. A 10× 0.5 NA air objective was used (TL10X-2P; ThorLabs). For optogenetic stimulation of Chrimson via the implanted lens, a 617 nm LED (M617L3; ThorLabs) controlled by an LED driver (T-Cube; ThorLabs) was used (5 – 10 mW at the objective face). One of the stimuli (“Cue A”) was paired with photostimulation of VTADA→BA axon terminals, which occurred 200 ms after the visual stimulus offset, and the second (“Cue B”) was not paired with any outcome (Figure S1C ).
Tl10x 2p
Manufactured by Thorlabs
The TL10X-2P is a 10X Galilean beam expander designed for use with collimated laser beams. It consists of a pair of plano-convex lenses that expand the beam diameter by a factor of 10 while maintaining collimation.
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3 protocols using tl10x 2p
1
Two-Photon Imaging with Optogenetic Stimulation
2
Two-Photon Calcium Imaging in Behaving Mice
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Two-photon calcium imaging was performed with a custom-built resonant-scanning multi-area two-photon microscope with a 10x/0.5NA, 7.77mm WD air objective (TL10X-2P, Thorlabs) using custom-written Scope software33 (link). A 31.25 MHz 1040 nm fiber laser (Spark Lasers) was used for RCaMP1.07 imaging. Simultaneous imaging at 32.6 Hz frame rate was performed of two imaging planes in L2/3 separated 50 µm in depth. For GRAB-Ach3.0 or GFP imaging, a single area at 32.6 Hz frame rate was acquired using an 80MHz ti:sapphire laser (Mai Tai HP DeepSee, Spectra Physics) tuned to 950 nm. Average power of each beam at the sample was 50–90mW. Imaging was performed during head-fixed task behavior or during passive stimulation sessions in naïve animals using similar stimulus conditions as T5.
3
Multimodal Neuronal Activity Imaging
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Rats were anesthetized with isoflurane (4% for induction, 0.5% during recording) and sedated with Chlorprothixene Hydrochloride (intramuscular injection of 300µl of 1 mg/ml solution, Sigma) as was used previously in mice to achieve stable recordings with minimal side effects of isoflurane on neuronal activity [17, 21, 23, 27, 28, 38] . Rats were placed on a heating pad, and were restrained and head-fixed to a custom mount under a two-photon microscope (Bergamo II, Thorlabs). Fluorescence signal was recorded using a 950nm wavelength (50-150 mW, Insight X3, Spectra-Physics) using an 8KHz resonant scanner (Cambridge Technology) and GaAsP photomultiplier tube (Thorlabs PMT2101). We collected light from fields of view (FOVs) with 512x512 pixels at 30 frames per second and with a 16x 0.8NA objective (CFI75 LWD, Nikon) for comparing spontaneous activity between GCaMP6f-and jGCaMP7s-labeled neurons, or a 10x 0.5NA objective lens (TL-10X 2P, Thorlabs) for longitudinal activity recording experiments. The respective FOV size was 200x200 or 500x500 µm 2 .
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