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Background: Brain visual circuits are often studied in vivo by imaging Ca²⁺ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals.

New method: To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca²⁺ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials –VEPs) and 3D acousto-optic two-photon (2P) population Ca²⁺ imaging in mouse primary visual cortex (V1).

Results: VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca²⁺ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable.

Comparison with existing methods: Masking or shielding the microscope are often used to minimize the contamination of Ca²⁺ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding.

Conclusions: Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca²⁺ imaging with electrophysiology and optogenetics.