Images were acquired using a custom-built two-photon laser-scanning microscope22 (link). A 20x water immersion microscope objective was used (W Plan-Apochromat 20x/1.0 DIC VIS-IR, Carl Zeiss AG, Feldbach, Schweiz). GCaMP2 and Texas Red were both excited at 900 nm with an InSight DeepSee laser (Newport Spectra Physics, Darmstadt, Germany) with a laser power between 10 and 30 mW. Fluorescence emission was detected with a GaAsP photomultiplier module (Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany) with a band-pass filter BrightLine HC 535/50 and a BrightLine 630/69 (LLC Semrock Inc., Rochester, USA). The two-photon laser-scanning microscope was controlled by a customized version of “ScanImage” (r3.8.1; Janelia Farm Research Campus, Ashburn VA, USA).
W plan apochromat 20x 1.0 dic vis ir
Manufactured by Zeiss
The Zeiss W Plan-Apochromat 20x/1.0 DIC VIS-IR is a high-performance objective lens designed for advanced microscopy applications. It features a numerical aperture of 1.0 and is optimized for use in both visible and infrared wavelength ranges.
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2 protocols using w plan apochromat 20x 1.0 dic vis ir
1
Two-Photon Imaging of Neuronal Calcium Dynamics
2
Two-Photon Microscopy for Cortical Imaging
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A custom-made two-photon laser scanning microscope 84 (link) equipped with a two-photon laser with <120 fs temporal pulse width (InSight DeepSee Dual; Spectra-Physics) with a 20x water immersion objective (W Plan-Apochromat 20x/1.0 DIC VIS-IR; Zeiss) was used for image acquisition. Excitation and emission beam paths were separated by a dichroic mirror (F73-825; AHF Analysentechnik). A dichroic mirror at 506nm (F38-506; AHF Analysentechnik) and 560nm (F38-560; AHF Analysentechnik) separated the emission light beam. The emission light was focused on the photomultiplier (H9305-03, Hamamatsu) by two lenses (LA1050-A1 and AL5040-A2; Thorlabs). We used emission filters for blue (F39-477; AHF Analysentechnik), green and yellow (F37-545; AHF Analysentechnik) and red (F39-608; AHF Analysentechnik) for multi-color imaging. Data was acquired from the somatosensory cortex 100-200 µm below the cortical surface. Anatomical imaging was performed at a resolution of 512×512 pixels at a frequency of 0.74 Hz. Emission signals were acquired at 11.84 Hz and with a 128×256-pixel resolution.
ScanImage 85 (link) and custom-written LabVIEW software (National Instruments) were used for image control and data acquisition.
ScanImage 85 (link) and custom-written LabVIEW software (National Instruments) were used for image control and data acquisition.
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