Raman spectroscopy returns chemical composition information of a sample.   It has the following advantages for studying growing and differentiating living systems: (i) it is non-destructive to the sample, (ii) analysis times are on the order of seconds, and (iii) there is no direct contact with the sample.  Raman spectra of living systems are convoluted and are composed of molecular signatures from the thousands of compounds comprising the system.  Here, we describe how multivariate statistics with discriminant analysis can (i) deconvolute Raman spectra into chemical composition information and (ii) be used to track differentiation of cells, tissues, and organs in near real-time.  This new approach is available as freely distributed stand-alone software and has been applied to the following case studies.  First, Raman spectroscopy was used to study the response of E. coli cultures to toxic 1-butanol exposure. Raman spectra were correlated with standard methods of analysis (e.g., GC-MS, fluorescence anisotropy) to identify Raman bands indicative of (i) changes in fatty acid chain length, saturation, and packing orientation; (ii) membrane fluidity; (iii) total protein content; and (iv) up-regulation of metabolic programs.  Next, E. coli cultures were exposed to different 4-carbon chain alcohols (i.e., 1-butanol, 2-butanol, isobutanol, tert-butanol, butanediol).  Significantly different toxicity responses were observed and elucidated.  In another case study, the health of an ex vivo perfused porcine liver was assessed.  Raman analysis was consistent with pathology results, suggesting the methodology has promise for determining when a preserved organ is no longer viable for transplant.  Finally, a new technology called �peptide-guided surface enhanced Raman scattering� (pg-SERS) will be introduced.  Using custom designed peptides covalently bound to silver nanoparticles, Raman spectra have been obtained from the sub-cellular level, and we have demonstrated the ability to target specific organelles or other sub-cellular locations (i.e., inside or outside of a cell membrane).

Raman spectroscopy for near real-time analysis of phenotypic changes in cells, tissues, and organs

247th National Meeting (2014)

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