Abstract
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.
Highlights
Optical microscopy provides a way to investigate bio-samples with high spatial resolution, low invasiveness, and high speed
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
Summary
Optical microscopy provides a way to investigate bio-samples with high spatial resolution, low invasiveness, and high speed. In order to acquire the spatial and spectral information simultaneously, different configurations based on the point-scanning system have been proposed and demonstrated. The other method uses a tunable filter device, such as a liquid crystal tunable filter (LCTF) or acoustic-optical tunable filter (AOTF) Such systems can achieve higher spectral resolutions; only one excitation wavelength is required [14,15]. CCD-based systems can provide more recording bands, that is, higher spectral resolution, thanks to the smaller pixel size. To enhance the signal-to-noise ratio (SNR), the exposure time of the CCD is set to be nT, where n is an integer number, in order to record the complete x-λ information at a single yposition. Limited by the CCD frame rate, the minimum acquisition time of this system is about 100 s for a full-size data stack. Drops of the diluted mixture were placed between the slide and cover glass for imaging
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