Fig. 1

a A multispectral fluorescent dataset is acquired using a confocal instrument in spectral mode (32 channels). Here we show a Tg(ubi:Zebrabow)³⁴ dataset where cells contain a stochastic combination of cyan, yellow, and red fluorescent proteins. b Average spectra within six regions of interest (colored boxes in a) show the level of overlap resulting in the sample. c Standard multispectral visualization approaches have limited contrast for spectrally similar fluorescence. d Spectra for each voxel within the dataset are represented as a two-dimensional histogram of their Sine and Cosine Fourier coefficients S and G, known as the phasor plot. e Spatially lossless spectral denoising is performed in phasor space to improve signal²⁹. f SEER provides a choice of several color reference maps that encode positions on the phasor into predetermined color palettes. The reference map used here (magenta selection) is designed to enhance smaller spectral differences in the dataset. g Multiple contrast modalities allow for improved visualization of data based on the phasor spectra distribution, focusing the reference map on the most frequent spectrum, on the statistical spectral center of mass of the data (magenta selection), or scaling the map to the distribution. h Color is assigned to the image utilizing the chosen SEER reference map and contrast modality. i Nearly indistinguishable spectra are depicted with improved contrast, while more separated spectra are still rendered distinctly.