The 2-photon calcium imaging setup was identical to a previously published design15 (link). Two-photon illumination was achieved with a Ti:Sapphire laser (Chameleon Vision II, Coherent) operating at 920nm. Fluorescence was acquired using a 40× 0.8 NA objective (LUMPLFLN40X/W, Olympus) and GaAsP PMTs (H10770PA-40, Hamamatsu) after passing through a dichroic (FF670-SDi01, Semrock), an IR filter (FF01–720sp, Semrock), reflected by a second dichroic (FF562-Di03, Semrock) and passing through a final bandpass filter (FF01–520/60, Semrock). The PMT output signal was amplified (Variable High Speed Current Amplifier; #59–179, Edmund Optics) and digitized (PXIe-7961R FlexRIO, National Instrument). The microscope was controlled by ScanImage (Vidrio Technologies) software using additional analog output units (PXIe-6341, National Instruments) for the laser power control and the scanners control. Double-distilled water was used as the immersion medium for the objective. Average beam power measured at the front of the objective was 60–160 mW. The region between the objective and imaging site was shielded from external sources of light using a black rubber tube. Horizontal scans of the laser were achieved using a resonant galvanometer (Thorlabs). Typical fields of view measured approximately 500×500 μm, and data was acquired at 30Hz.
Resonant galvanometer
Manufactured by Thorlabs
Sourced in United States
The Resonant Galvanometer is a precision electromechanical device designed to detect and measure oscillating or alternating currents. It operates on the principle of a resonant circuit, allowing for highly sensitive and accurate current measurements across a range of frequencies.
Automatically generated - may contain errors
Lab products found in correlation
H10770pa 40, by Hamamatsu Photonics (2 mentions)
Ff01 720 sp, by IDEX Corporation (1 mentions)
Pxie 6341, by National Instruments (1 mentions)
Ff670 sdi01, by IDEX Corporation (1 mentions)
Ff562 di03, by IDEX Corporation (1 mentions)
Lumplfln 40xw, by Olympus (1 mentions)
Gaasp photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Matlab, by MathWorks (1 mentions)
3 protocols using resonant galvanometer
1
Two-Photon Calcium Imaging Setup
2
Two-Photon Imaging of Neuronal Activity
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We used a laser-scanning two-photon microscope to detect the GCaMP6s fluorescence signal. The signal was detected using a bandpass filter (No.525/50; Semrock, New York, USA) and GaAsP photomultiplier tubes (No.10770PB-40; Hamamatsu). We used a resonant galvanometer (Thorlabs, New Jersey, USA; 16 kHz line rate, bidirectional) for horizontal scanning, which was sealed in an optical window to reduce noise levels to below 30 dB. The entire microscope was enclosed in a double-walled sound-attenuation box to ensure that the internal noise level was below 30 dB during imaging. Images were obtained using the ScanImage software r3.0 (http://scanimage.org , accessed on 11 December 2022). Calcium transients were imaged in the cortex 200–300 μm beneath the pial surface, using 512 × 512 (soma) or 1024 × 1024 (spine) pixel images. We recorded the spontaneous neuronal activity in the mice for 5 min once they had reached a stable state of anesthesia, approximately 20 min after injection. The resolution of the dendritic spines was 0.3 μm per pixel, while the resolution of the cell bodies was 0.8 μm per pixel [9 (link)].
3
Two-Photon Microscopy for Imaging
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We performed imaging with a custom two-photon microscope controlled with the Scan Image software (Vidrio) running in MATLAB (Mathworks). Laser illumination was provided by a Ti:Sapphire laser (Chameleon Vision II, Coherent) operating at 920 nm. We used a long working distance air immersion objective lens (20×/0.6NA/13 mm WD, Edmunds optics) and a GaAsP photomultiplier tubes (H10770PA-40, Hamamatsu). The beam power was modulated by a Pockels cell (350-80 LA BIC −02 Conoptics) and the power used for imaging, measured at the front of the objective, was 150–200 mW. Horizontal scans of the laser were achieved using a resonant galvanometer (Thorlabs). Typical fields of view measured ~600 × 600 μm and data were acquired at 30 Hz. The measured lateral and axial resolution of microscope given by the FWHM of the point spread function was 1.2 and 9.1 μm.
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