Cutrale, F. | Search bioRxiv

bioRxiv2025

Transforming Hyperspectral Data into Insight: The DREAM Approach for Pathology

Quantitative pathology remains limited due to the need for chemical staining and subjective interpretation of tissue features. Autofluorescence imaging offers a label-free alternative: however, high-dimensional excitation-emission datasets pose challenges for visualization and reproducible analysis. Here, we present Dimensionality Reduction for Enhanced Autofluorescence Microscopy (DREAM), a method that condenses multi-excitation emission spectra into a compact, information-rich format using phasor-based tools. Applied to unstained esophageal tissue samples, DREAM enables high-contrast visualizations that distinguish key histological structures without the need for exogenous labeling. Quantitative assessments across multiple datasets show DREAM improves colorfulness, sharpness, and consistency over single-laser acquisitions, supporting its potential to advance objective, label-free diagnostics through enhanced spectral visualization.

biophysics↗

bioRxiv2022

FRaeppli, a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish

Visualizing cell shapes, interactions and lineages of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated tracking cells in in vivo and mapping neuronal connectivity. Nevertheless, integrating multi-fluorophore information into the context of developing tissues in zebrafish is challenging given their cytoplasmic localization and spectral incompatibility with commonly used fluorescent markers. Here, we developed FRaeppli (Fish-Raeppli) expressing bright membrane-or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. By tailoring hyperspectral protocols for time-efficient acquisition, we demonstrate FRaepplis suitability for live imaging of complex internal organs, like the liver. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting shared developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and tissue lineages at single-cell resolution in zebrafish.

developmental biology↗