Abstract
Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz., background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LDs) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.
Highlights
Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling
Six different HuH7 cells were analyzed in the present study: three cells from each feed condition
multivariate curve resolution (MCR) components 1, 2, and 3 (Fig. 1d) are lipid spectra and we have assigned them as follows
Summary
Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. A study of the distribution of several different molecules inside LDs would require each of them to be selectively labeled In such efforts, it is necessary to avoid lager molecular structural alterations of the probe-molecule such that the biological functional equivalence of the labeled species is largely unaffected compared to its natural analogue. A critical evaluation of biological behavior of labeled and native molecules inside a cell is very important in the light of extensive use molecular labeling methodology in molecular biology and medicine. This would require selective detection of these derivatives when they are simultaneously present inside the same cell. Linear Raman imaging technique has often been criticized for the inferior signal strength compared to the nonlinear analogues such as coherent anti-stokes
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