Hyperspectral Imaging in a Single Plane Illumination Microscope by Spectral Phasor Analysis with Sine-Cosine Transmission Filters

Abstract

The combination of hyperspectral imaging and single plane illumination microscopy (SPIM) is a powerful approach to address large samples in a multidimensional fashion. We have developed an ultrafast phasor-based hyperspectral imaging method using sine/cosine interference filters on the side-SPIM microscope. This instrumentation is 10-100 times faster than regular hyperspectral imaging in scanning confocal, while maintaining resolution and achieving high photon recovery. We have tested this new method in Spectrum of Fates (SoFa) zebrafish lines, in which the expression of different fluorescent proteins is driven from promoters of a set of transcription factors differentially expressed in the developing retina: Crx:gapCFP, Ptf1a:cytGFP, and Atoh7:gapRFP. Thus, SoFa is an attractive model with several cell types co-expressing more than one FP, so no simple “single color” pattern was expected. SoFa zebrafish embryos were fixed at 72 hours post fertilization, nucleic acids stained with SYTOX Orange (SO) to label the nucleus and imaged by 4D (x,y,z,λ,) light sheet microscopy with the sine/cosine filter method. Using the reciprocity principle of the phasor transformation we were able to select image regions corresponding to the layer organization of the zebrafish retina at this embryonic stage and build the corresponding phasor histograms. Our results showed an excellent performance of the sine/cosine filter method in combination with SPIM to identify and quantify each of the fluorescent components in the different cell types, corresponding to their well characterized expression. Hence, this work present a new method simplifies hyperspectral imaging in a SPIM microscope, opening new perspectives for 5D (x,y,z,λ,t) imaging of complex live biological samples.