Protocol detail

Two-Photon Microscopy for Glutamate Imaging

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We used a MOM-type two-photon microscope (designed by W. Denk, MPI, Heidelberg; purchased from Sutter Instruments/Science Products^{113 (link)}). The system was equipped with a mode-locked Ti:Sapphire laser tuned to 927 nm (MaiTai-HP DeepSee, Newport Spectra-Physics), two fluorescence detection channels for iGluSnFR/GCaMP6f (HQ 510/84, AHF/Chroma) and SR101 (HQ 610/75, AHF), and a water immersion objective (W Plan-Apochromat × 20 /1.0 DIC M27, Zeiss). For image acquisition, we used custom-made software (ScanM by M. Müller and T.E.) running under IGOR Pro 6.37 for Windows (Wavemetrics), taking time-lapsed 64 × 64 pixel image scans (at 9.766 Hz) or 128 × 32 pixel image scans (at 15.625 Hz). For vertical glutamate imaging in the IPL, we recorded time-lapsed 64 × 56 pixel image scans (at 11.16 Hz) using an electrically tunable lens (ETL; for details, see ref. 61 (link)).

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Strauss S., Korympidou M.M., Ran Y., Franke K., Schubert T., Baden T., Berens P., Euler T, & Vlasits A.L. (2022). Center-surround interactions underlie bipolar cell motion sensitivity in the mouse retina. Nature Communications, 13, 5574.

Publication 2022

MaiTai-HP DeepSee W Plan-Apochromat × 20 /1.0 DIC M27 IGOR Pro 6.37

Electrically Fluorescence Glutamate Immersion Lens Microscope Sapphire Scans

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Variable analysis

independent variables

Laser tuned to 927 nm

dependent variables

Fluorescence of iGluSnFR/GCaMP6f
Fluorescence of SR101
Vertical glutamate imaging in the IPL

control variables

Water immersion objective (W Plan-Apochromat × 20 /1.0 DIC M27, Zeiss)
Custom-made software (ScanM by M. Müller and T.E.) running under IGOR Pro 6.37 for Windows (Wavemetrics)
Time-lapsed 64 × 64 pixel image scans (at 9.766 Hz) or 128 × 32 pixel image scans (at 15.625 Hz)
Time-lapsed 64 × 56 pixel image scans (at 11.16 Hz) using an electrically tunable lens (ETL)

controls

No positive or negative controls were explicitly mentioned.

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