Analysis of Single Bacterial Spores by Micro-Raman Spectroscopy

The spectroscopic analysis of individual living cells in their native state provides a powerful tool for the investigation of complex biological systems. Micro-Raman spectroscopy, in which confocal microscopy is combined with Raman spectroscopy, offers a promising route to achieving this, because it provides a means to study individual cells and cellular components.1–9 Here we describe the analysis of individual bacterial endospores from four species in the genus Bacillus by micro-Raman spectroscopy. Previous Raman studies on Bacillus spores resulted in spectra with strong scattering from calcium dipicolinate (CaDPA), which is the calcium chelate of dipicolinic acid (2,6-pyridinedicarboxylic acid, DPA); however, these earlier studies were conducted on concentrated samples of spores.10–14 By using micro-Raman spectroscopy, we demonstrate the ability to obtain similar information from individual spores. The Raman spectra for most spores studied were dominated by scattering from CaDPA, although Raman scattering assignable to protein bands and to phenylalanine was also observed. Approximately 4% of the spores analyzed did not exhibit Raman intensity from CaDPA, possibly due to incomplete sporulation. The results presented indicate that micro-Raman spectroscopy is a promising technique for in-

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