Non-de-scanned parallel recording two-photon hyperspectral microscopy with high spectral and spatial resolution.

A two-photon hyperspectral microscope with non-de-scanned geometry and parallel recording scheme was constructed which had a low dwell time, high spectral resolution, and high spatial resolution. The dwell time was effectively decreased to reduce photodamage through parallel recording, while the non-de-scanned geometry led to a considerable reduction in the signal loss and spectrum distortion. Two experiments were performed to show that this system can solve crosstalk issues and spectrally resolve the intrinsic fluorophores in optically-thick tissues without staining and sectioning.