In vivo Two-Photon Calcium Imaging

In vivo two-photon calcium imaging^{95 (link)} was performed with a customized commercially available Bergamo II (Thorlabs) two-photon laser scanning microscope^{96 (link)} using a pulsed femtosecond Ti:Sapphire laser (Mai Tai HP Deep See, Spectra-Physics) and controlled by ScanImage 4 (ref. ^{97 (link)}). The calcium indicator GCaMP6m^{98 (link)} and the structural marker mRuby2 (ref. ^{99 (link)}) were both excited with a wavelength of 940 nm. Emitted photons were filtered for reflected laser light (720/25 short-pass filter), spectrally separated using a dichroic beamsplitter (FF560) and two band-pass filters (500–550 nm for GCaMP6m; 572–642 nm for mRuby2) and detected using two GaAsP photomultiplier tubes. Laser power was kept between 18 and 35 mW, depending on the depth of imaging and the quality of the chronic window. Images were acquired from two alternating planes, 40 μm apart, using a ×16 0.8-NA objective (Nikon) mounted on a piezoelectric stepper (Physik Instrumente). The xy image dimensions were 325 × 250 μm (512 × 512 pixels), and each image plane was acquired at a rate of ~15 Hz (total frame rate of ~30 Hz).

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Goltstein P.M., Reinert S., Bonhoeffer T, & Hübener M. (2021). Mouse visual cortex areas represent perceptual and semantic features of learned visual categories. Nature Neuroscience, 24(10), 1441-1451.

Publication 2021

Bergamo II Deep See ×16 0.8-NA objective

Calcium Frame Light Sapphire

Corresponding Organization : Max Planck Institute of Neurobiology

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Variable analysis

independent variables

Wavelength of laser used to excite the calcium indicator GCaMP6m and the structural marker mRuby2 (940 nm)

dependent variables

Calcium dynamics measured using the calcium indicator GCaMP6m
Structural information provided by the marker mRuby2

control variables

Laser power (kept between 18 and 35 mW)
Imaging depth
Quality of the chronic window
Xy image dimensions (325 × 250 μm, 512 × 512 pixels)
Imaging frame rate (~15 Hz per plane, total ~30 Hz)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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