Metabolically activated Raman contrast enhanced by optical clearing for label-assisted mapping of tissue pathology

Abstract

Spatial assessment of tissue metabolic activity may provide valuable information for identifying pathological regions, including ischemic lesions and tumor nodules. In this study, we propose a Raman spectroscopic approach based on metabolically activated contrast produced by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan in viable tissues.
Fresh organotypic tissue samples were incubated with MTT for a short period and subsequently examined by Raman spectroscopy. Experimental measurements demonstrated that MTT-derived formazan generated a strong and well-defined Raman spectrum characterized by a distinct spectral fingerprint within the tissue. Several characteristic formazan bands were located in spectral regions with minimal overlap with intrinsic tissue Raman bands, enabling selective detection and providing the basis for spatial Raman mapping.
To improve the detectability of formazan in optically heterogeneous tissues, optical clearing was incorporated into the protocol. Ex vivo tissue samples were treated with a glycerol/DMSO/water mixture at a volume ratio of 70:5:25. Optical clearing increased the measured intensity of the selected formazan-associated Raman bands, indicating improved recovery of the Raman signal from the tissue.
The proposed approach combines short-term metabolic labeling, Raman spectroscopic mapping, and optical clearing. We hypothesize that differences in the rate and spatial distribution of MTT reduction allow discrimination between normal and pathological regions within the same organ. Our experiments evaluate this approach in kidney and brain tissues containing ischemic lesions or tumor nodules and assess its potential for spatially resolved ex vivo metabolic imaging.

Speaker

Olga Gusliakova
Saratov State University
Russian Federation

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