Effects of ethanol,formaldehyde, and gentle heat fixation in confocal resonance Raman microscopy of purple nonsulfur bacteria |
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Authors: | Ann‐Kathrin Kniggendorf Tobias William Gaul Merve Meinhardt‐Wollweber |
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Affiliation: | Institute of Biophysics, Gottfried Wilhelm Leibniz University of Hannover, Herrenhaeuser Str. 2, 30419 Hannover, Germany |
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Abstract: | Resonance Raman microscopy is well suited to examine living bacterial samples without further preparation. Therefore, comparatively little thought has been given to its compatibility with common fixation methods. However, fixation of cell samples is a very important tool in the microbiological sciences, allowing the preservation of samples in a specific condition for further examination, future measurements, transport, or later reference. We examined the effects of three common fixatives—ethanol, formaldehyde solution, and gentle heat—on the resonant Raman spectrum of three generic bacteria species, Rhodobacter sphaeroides DSM 158T, Rhodopseudomonas palustris DSM 123T, and Rhodospirillum rubrum DSM 467T, holding carotenoid‐ and heme‐chromophores in confocal Raman microscopy. In addition, we analyzed the effect of poly‐L ‐lysine coating of microscope slides, widely used for mounting biological and medical samples, on subsequent confocal Raman measurements of native and fixed samples. The results indicate that ethanol is preferable to formaldehyde as fixative if applied for less than 24 h, whereas heat fixation has a strong, detrimental effect on the resonant Raman spectrum of bacteria. Formaldehyde fixation excels at fixation times above 24 h, but causes an overall reduction in signal intensity. Poly‐L ‐lysine coating has no discernable effect on the Raman spectra of samples fixed with ethanol or heat, but it further decreases the signal intensity, especially at higher wavenumbers, in the spectra of samples fixed with formaldehyde. Microsc. Res. Tech. 74:177–183, 2011. © 2010 Wiley‐Liss, Inc. |
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Keywords: | resonance Raman spectroscopy fixation confocal laser microscopy poly‐L‐lysine purple non‐sulfur bacteria |
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