Raman spectroscopy of microbial pigments

Abstract

Raman spectroscopy is a rapid nondestructive technique providing spectroscopic and structural information on both organic and inorganic molecular compounds. Extensive applications for the method in the characterization of pigments have been found. Due to the high sensitivity of Raman spectroscopy for the detection of chlorophylls, carotenoids, scytonemin, and a range of other pigments found in the microbial world, it is an excellent technique to monitor the presence of such pigments, both in pure cultures and in environmental samples. Miniaturized portable handheld instruments are available; these instruments can be used to detect pigments in microbiological samples of different types and origins under field conditions.

FIG 1

(a) Conventional confocal Raman microspectrometer. The laser irradiates the object selected in the optical microscope (laser spot ≈ 2 μm), the scattered light from the sample is collected by the optics of the microscope passing through holographic filters, pinhole, and monochromator to be detected by a charge-coupled device (CCD). (b) Handheld Raman spectrometer allows direct measurements on outcrops (cyanobacteria on siliciclastic rocks at Albertov, Prague, Czech Republic). The rugged probe head permits laser illumination of the object and collection of the scattered light.

FIG 2

Examples of microbiological application of Raman spectroscopy using different instrumentation. (a to c) Raman spectra obtained using a laboratory Raman microspectrometer. Raman spectra of salinixanthin from a culture of Salinibacter ruber (a) (532-nm wavelength; image modified from reference 54), of flexirubin from a culture of Flavobacterium johnsonae (b) (532-nm wavelength; image modified from reference 57), and of chlorophyll (□), carotenoid (▼), and scytonemin (*) from a native gypsum crust (Yungay, Atacama, Chile) (c) (785-nm wavelength; image modified from reference 69). (d) Raman spectrum collected using a handheld Raman spectrometer of a spirilloxanthin-like carotenoid of the red layer of the bottom gypsum crust from a saltern at Eilat (Israel) colonized by Halochromatium-like microorganisms (532-nm wavelength; image modified from reference 117). (e) Example of Raman mapping–distribution of carotenoids (astaxanthin, β-carotene, chlorophyll in the frame of flagellated motile cell obtained using a confocal Raman spectrometer (532-nm wavelength; image modified from reference , which was published under a Creative Commons agreement). The intensities within the concentration map represent relative component concentrations. Bars, 10 μm.