Visual stimulation with blue wavelength light drives V1 effectively eliminating stray light contamination during two-photon calcium imaging
BACKGROUNDBrain 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 METHODTo overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicators 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). RESULTSVEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging 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 METHODSMasking 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 without affecting V1 physiological responsiveness. CONCLUSIONSOur results show that the selected blue light stimuli evoke physiological 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. HighlightsO_LIWhite and blue light trigger VEPs with similar amplitudes and latencies in mouse V1 C_LIO_LIBlue-and white-responding neurons are two largely overlapping neuronal populations C_LIO_LIBlue and white evoke Ca2+ responses similar in magnitude and latency C_LIO_LIBlue and white evoke Ca2+ responses similar in orientation/direction selectivity C_LIO_LIBlue stimuli could be an alternative to white ones in behavior and opto-physiological tests C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/433182v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@66c0a2org.highwire.dtl.DTLVardef@1f0cce9org.highwire.dtl.DTLVardef@1ce97borg.highwire.dtl.DTLVardef@24a3f1_HPS_FORMAT_FIGEXP M_FIG C_FIG