Orthogonally-polarized excitation for improved two-photon and second-harmonic-generation microscopy, applied to neurotransmitter imaging with GPCR-based sensors.

Abstract

Fluorescent proteins are excited by light that is polarized parallel to the dipole axis of the chromophore. In two-photon microscopy, polarized light is used for excitation. Here we reveal surprisingly strong polarization sensitivity in a class of genetically encoded, GPCR-based neurotransmitter sensors. In tubular structures such as dendrites, this effect led to a complete loss of membrane signal in dendrites running parallel to the polarization direction of the excitation beam. To reduce the sensitivity to dendritic orientation, we designed an optical device that generates interleaved pulse trains of orthogonal polarization. The passive device, which we inserted in the beam path of an existing two-photon microscope, removed the strong direction bias from fluorescence and second-harmonic (SHG) images. We conclude that for optical measurements of transmitter concentration with GPCR-based sensors, orthogonally polarized excitation is essential.