To begin, affix a two-axis goniometer with a titanium plate to the stage plate for XY positioning under the microscope. Activate the scanning software, set pixels to 512 by 512 bidirectional to on using a 20X objective lens. After performing the cranial window surgery, place the pup expressing GCaMP on one side of the hemisphere on the heating pad.
Using screws, secure the head mount titanium bar to the titanium plate. Then adjust the window angle horizontally with the goniometer. Illuminate the brain surface with the backlight and select the imaging area using XY positioning with a 5X objective lens.
Under a 20X water immersion lens, administer eyedrops to cranial window. After disabling the backlight, scan the brain surface in one photon mode. Increase the laser intensity to visualize the green autofluorescence from the glass and brain surface.
Cover the microscope to prevent external light interference. Start the scanning software to two photon mode for imaging. Then calibrate the laser power and detector gain to optimally visualize GCaMP signals.
Locate the depth in an imaging area populated with many GCaMP-expressing neurons and start time-lapse imaging to capture GCaMP signals. Two-photon microscopy revealed layer four neuron activities in the barrel cortex of a postnatal day six pup with green fluorescence. The green GCaMP was more prominent, resulting in signal leakage into the red channel and identification of 14 regions of interest.
