Effects of ethanol, formaldehyde, and gentle heat fixation in confocal resonance Raman microscopy of purple nonsulfur bacteria

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 158(T), Rhodopseudomonas palustris DSM 123(T), and Rhodospirillum rubrum DSM 467(T), 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.

Raman Spectroscopy of Microbial Pigments

“…A feature of medium intensity occurs at 1,008 cm Ϫ1 ( 3 ), corresponding to the in-plane rocking modes of the CH 3 groups attached to the polyene chain. The wavenumber positions of both 1 and 2 bands depend on the length of the polyene chain (the number of conjugated double bonds) (37)(38)(39)(40). The shift in band position is much more pronounced in the case of the 1 band; a longer conjugated polyene chain causes a shift in the 1 band to lower wavenumber positions and vice versa (37).…”

Intracellular SERS Nanoprobes For Distinction Of Different Neuronal Cell Types

“…Fixation with formaldehyde is widely used for biological sample preparation and has been shown to have no discernible effects on acquired spectra. 39,40 …”

Evaluation of whole cell fixation methods for the analysis of nanoscale surface features of Yersinia pestis KIM

“…The mass spectrometry signature quality improved using methanol treatment, showing the preservation of biomacromolecules on cell surfaces (Qian et al ., ). In a related study of the effect of three common inactivation methods (heat, formaldehyde, alcohol) on the confocal resonance Raman spectra of purple nonsulfur bacteria, ethanol performed better than formaldehyde, whereas heat treatment had a strong, adverse effect on the resonant Raman spectrum (Kniggendorf et al ., ). Overall, compared to formaldehyde, there are several advantages of using alcohols: (i) lower toxicity and easy handling, (ii) limited crosslinking, while formaldehyde crosslinking masks available specific binding sites of antigens hampering immunohistochemistry or bioactivity, (iii) superior ultrastructure in comparison to formaldehyde treated cells (Dapson, ; van Essen et al ., ; Sjostedt et al ., ), (iv) permit immunological staining for most antibodies if needed downstream (van Essen et al., ).…”