Changes in GCaMP6s fluorescence were imaged with a Two-photon Moveable Objective Microscope (Sutter) and a mode-locked Ti:sapphire laser (Chameleon Vision II, Coherent) at 950 nm through a 25 x (1.05 NA, Olympus) water immersion objective at 4.07 fps with 512 × 512 pixels resolution using Mscan software (Sutter). Images were obtained with a 535/50 and 610/75 nm band-pass emission filters for the green and red channels, respectively. Each field-of-view (FOV) contained 4–19 Ca2+ indicator filled VIPs or SOMs. Imaging data was analyzed using ImageJ and custom routines in MATLAB (see Supplemental Experimental Procedures).
Mscan software
Manufactured by Sutter Instruments
MScan software is a data acquisition and analysis tool developed by Sutter Instruments. It provides a user-friendly interface for recording and processing data from various laboratory instruments.
Automatically generated - may contain errors
3 protocols using mscan software
1
Two-photon Imaging of GCaMP6s Fluorescence
2
Two-Photon Imaging of Cerebral Vasculature
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Two-photon imaging was performed with a Sutter Moveable Objective Microscope. A MaiTai HP (Spectra-Physics, Santa Clara, California) laser tuned to 800 nm was used for fluorophore excitation. Before imaging, the mouse was briefly anesthetized with isoflurane (5% in oxygen), retro-orbitally injected with 50 μL of 70 kDa fluorescein-conjugated dextran (Sigma-Aldrich) prepared at a concentration of 5% (weight/volume) in sterile saline to label plasma, and then fixed on a spherical treadmill. Imaging was done with a 20X, 1.0 NA objective (Olympus, XLUMPFLN). Control of 2PLSM and data acquisition was accomplished using MScan software (Sutter Instruments). All imaging with the water-immersion lens was done with room temperature distilled water. Wide-field images were collected to generate vascular maps of the entire window for navigational purposes. High-resolution images of the vasculature were collected using a 500 μm by 500 μm field for measurement of capillary diameter. Capillary diameter was measured using ImageJ software. To measure RBC velocity and RBC spacing, line scan images were collected from individual capillaries. RBCs appeared as tilted dark shadows on a bright background due to the fluorescein-conjugated dextran contained in the blood plasma (Fig 6A
3
Calcium Imaging of Immobilized Fish
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The selected fish were immobilized in 1.7% low melting-temperature agarose (wt/vol, E3 medium) at the tip of a transparent plastic triangle stage (tip angle < 45%, was collected with a 20x/1.0 Zeiss objective using an emission dichroic with bandpass 500-550 nm. The image time series were recorded using MScan software (Sutter instruments). The calcium images acquisition rate was 2 fps at 512 × 512 pixel 2 and a magnification of 2 (0.44 × 0.44 μm 2 per pixel).
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