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.

A set of standard reference maps and their corresponding result on a Simulated Hyperspectral Test Chart (SHTC) designed to provide a gradient of spectral overlaps between spectra. a The Standard phasor plot with corresponding average grayscale image provides the positional information of the spectra on the phasor plot. The phasor position is associated to a color in the rendering according to a set of standard reference maps, each highlighting a different property of the dataset. b The angular map enhances spectral phase differences by linking color to changes in angle (in this case, with respect to origin). This map enhances changes in maximum emission wavelength, as phase position in the plot is most sensitive to this feature, and largely agnostic to changes in intensity. c The radial map, instead, focuses mainly on intensity changes, as a decrease in the signal-to-noise generally results in shifts towards the origin on the phasor plot. As a result, this map highlights spectral amplitude and magnitude, and is mostly insensitive to wavelength changes for the same spectrum. d The gradient ascent map enhances spectral differences, especially within the higher intensity regions in the specimen. This combination is achieved by adding a brightness component to the color palette. Darker hues are localized in the center of the map, where lower image intensities are plotted. e The gradient descent map improves the rendering of subtle differences in wavelength. Colorbars for b, c, d, e represent the main wavelength associated to one color in nanometers. f The tensor map provides insights in statistical changes of spectral populations in the image. This visualization acts as a spectral edge detection on the image and can simplify identification of spectrally different and infrequent areas of the sample such as the center of the SHTC. Colorbar represents the normalized relative gradient of counts.

For each SEER standard reference map design, four different modes can provide improved contrast during visualization. As a reference we use the gradient descent map applied on a Simulated Hyperspectral Test Chart (SHTC). a Standard mode is the standard map reference. It covers the entire phasor plot circle, centering on the origin and anchoring on the circumference. The color palette is constant across samples, simplifying spectral comparisons between datasets. b Scaled mode adapts the gradient descent map range to the values of the dataset, effectively performing a linear contrast stretching. In this process the extremities of the map are scaled to wrap around the phasor representation of the viewed dataset, resulting in the largest shift in the color palette for the phase and modulation range in a dataset. c Max morph mode shifts the map center to the maximum of the phasor histogram. The boundaries of the reference map are kept anchored to the phasor circle, while the colors inside the plot are warped. The maximum of the phasor plot represents the most frequent spectrum in the dataset. This visualization modality remaps the color palette with respect to the most recurring spectrum, allowing insights on the distribution of spectra inside the sample. d Mass morph mode, instead, uses the histogram counts to calculate a weighted average of the phasor coordinates and uses this color-frequency center of mass as a new center for the SEER map. The color palette now maximizes the palette color differences between spectra in the sample.

The sample was imaged using multispectral two-photon microscopy (740 nm excitation, 32 wavelength bins, 8.9 nm bandwidth, 410–695 nm detection) to collect the fluorescence of intrinsic molecules including folic acid, retinoids and NADH in its free and bound states. These intrinsic molecules have been used as reporters for metabolic activity in tissues by measuring their fluorescence lifetime, instead of wavelength, due to their closely overlapping emission spectra. This overlap increases the difficulty in distinguishing spectral changes when utilizing a (a) TrueColor image display (Zen Software, Zeiss, Germany). b The gradient descent morphed map shows differences between apical and basal layers, suggesting different metabolic activities of cells based on the distance from the tracheal airway. Cells on the apical and basal layer (dashed boxes) are rendered with distinct color groups. Colorbar represents the main wavelength associated to one color in nanometers. c The tensor map image provides an insight of statistics in the spectral dataset, associating image pixels’ colors with corresponding gradient of phasor counts for pixels with similar spectra. The spectral counts gradients in this sample highlights the presence of fibers and edges of single cells. Colorbar represents the normalized relative gradient of counts. d Average spectra for the cells in dashed boxes (1 and 2 in panel c) show a blue spectral shift in the direction of the apical layer. e Fluorescence Lifetime Image Microscopy (FLIM) of the sample, acquired using a frequency domain detector validates the interpretation from panel (b), Gradient descent map, where cells in the apical layer exhibit a more oxidative phosphorylation phenotype (longer lifetime in red) compared with cells in the basal layer (shorter lifetime in yellow) with a more glycolytic phenotype. The selections correspond to areas selected in phasor FLIM analysis (e, top left inset, red and yellow selections) based on the relative phasor coordinates of NAD+/NADH lifetimes³⁵.

Tg(fli1:mKO2) (pan-endothelial fluorescent protein label) zebrafish was imaged with intrinsic signal arising from the yolk and xanthophores (pigment cells). Live imaging was performed using a multispectral confocal (32 channels) fluorescence microscope with 488 nm excitation. The endothelial mKO2 signal is difficult to distinguish from intrinsic signals in a (a) maximum intensity projection TrueColor 32 channels Image display (Bitplane Imaris, Switzerland). The SEER angular map highlights changes in spectral phase, rendering them with different colors (reference map, bottom right of each panel). b Here we apply the angular map with scaled mode on the full volume. Previously indistinguishable spectral differences (boxes 1, 2, 3 in panel a) are now easy to visually separate. Colorbar represents the main wavelength associated to one color in nanometers. c–h Zoomed-in views of regions 1–3 (from a) visualized in TrueColor (c, e, g) and with SEER (d, f, h) highlight the differentiation of the pan-endothelial label (yellow) distinctly from pigment cells (magenta). The improved sensitivity of SEER further distinguishes different sources of autofluorescence arising from yolk (blue and cyan) and pigments.

Zebrafish embryo Tg(kdrl:eGFP); Gt(desmin-Citrine);Tg(ubiq:H2B-Cerulean) labeling, respectively, vasculature, muscle, and nuclei. Live imaging with a multispectral confocal microscope (32-channels) using 458 nm excitation. Single plane slices of the tiled volume are rendered with TrueColor and SEER maps. a TrueColor image display (Zen, Zeiss, Germany). b Angular map in center of mass morph mode improves contrast by distinguishable colors. The resulting visualization enhances the spatial localization of fluorophores in the sample. c Gradient descent map in max morph mode centers the color palette on the most frequent spectrum in the sample, highlighting the spectral changes relative to it. In this sample, the presence of skin pigment cells (green) is enhanced. 3D visualization of SEER maintains these enhancement properties. Colorbars represent the main wavelength associated to one color in nanometers. Here we show (d, e, f) TrueColor 32 channels Maximum Intensity Projections (MIP) of different sections of the specimen rendered in TrueColor, angular map center of mass mode and gradient descent max mode. The selected views highlight SEER’s performance in the d overview of somites, e zoom-in of somite boundary, and f lateral view of vascular system.

Maximum intensity projection renderings of Tg(ubi:Zebrabow) muscle³⁴ acquired live in multispectral confocal mode with 458 nm excitation. a The elicited signal (e.g., white arrows) is difficult to interpret in the TrueColor Image display (Zen Software, Zeiss, Germany). b Discerning spectral differences is increasingly simpler with gradient descent map scaled to intensities, while compromising on the brightness of the image. c Gradient descent and d gradient ascent RGB masks in scale mode show the color values assigned to each pixel and greatly improve the visual separation of recombined CFP, YFP, and RFP labels. Colorbars represent the main wavelength associated to one color in nanometers.