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Environment-specific spectral modeling: A new tool for the analysis of biological specimens.

5 Jun | By Biophotonics.World
Environment-specific spectral modeling: A new tool for the analysis of biological specimens.
By: Giovanni Valbusa, Martina Capozza, Chiara Brioschi

The recent discovery of fluorescent dyes for improving pathological tissues identification has highlighted the need of robust methods for performance validation especially in the field of Fluorescence Guided Surgery (FGS). Optical imaging of excised tissue samples is the reference tool to validate the association between dyes localization and the underlying histology in a controlled environment. Spectral unmixing may improve the validation process discriminating exogenous from endogenous signal. The existing spectral unmixing methods are effective in calculating dyes contributions to fluorescence intensity but they do not weight on spectral variations related to chemical environment that usually affect histopathology samples and biological specimens. 

The analysis of chemical environment-related effects on fluorescence spectra is normally carried out using spectrofluorometers with high spectral resolutions (about 1 nm) and dedicated in vitro experiments on fluorophores solutions with variable physicochemical properties. Optical imaging devices typically used for the analysis of tissue samples, conversely, have limited spectral resolution (about 20-30 nm). Moreover, the complex chemical environment of tissue samples cannot be reproduced to provide reference pure spectra. We have described an innovative spectral modeling approach that weights the spectral shifts associated with changes in chemical environment. 

Essentially, the method CASUM (Chemical Aware Spectral Unmixing) is a variation of linear unmixing, the most commonly used spectral unmixing method, but differently from linear unmixing, it takes into account spectra dependency on chemical environment. 

In the proof-of-concept study reported in the paper, the method is applied to formalin-fixed paraffin embedded (FFPE) tissues, obtained from a FGS study carried out on oncologic canine patients that received a tumor-specific fluorescent contrast agent (DA364). Here, the hypothesis that DA364 spectral shift is induced by FFPE blocks viscosity and/or dye interactions with the biological matrix is investigated. With the development of CASUM, the systematic unmixing biases, initially attributed to endogenous autofluorescence sources or spectral coloring, are eventually explained by DA364 spectral shift. CASUM is robust against spectral shift variations, its application leads to unbiased estimates of the spectral weights, as demonstrated by numerical simulations. Moreover, CASUM is able to extract from spectral images information related to the chemical environment, through the estimation of spectral shift parameters. Spectral shifts values computed pixel-wise from spectral images are used to display additional information with potential diagnostic value. 

In conclusion, CASUM is a tool to study fluorescent dyes interaction with tissues, it exploits fluorophores spectral sensitivity to chemical environment and it can be used with low resolution imaging devices, generally available in biology labs. 

Since the interest in chemical environment sensitivity has been widely reported in literature, CASUM can be considered a tool for studying biological solvation systems using low resolution devices. Original article Valbusa G, Capozza M, Brioschi C, Blasi F, Ghiani S, Maiocchi A. Environment-specific spectral modeling: A new tool for the analysis of biological specimens. J Biophotonics. 2019 Mar;12(3):e201800217.


Authors: Giovanni Valbusa, Martina Capozza, Chiara Brioschi


Methods and Techniques: Fluorescence spectroscopy

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