Raman spectroscopic signatures of carotenoids and polyenes enable label-free visualization of microbial distributions within pink biofilms

Abstract

Pink biofilms are multispecies microbial communities that are commonly found in moist household environments. the development of this pink stain is problematic from an aesthetic point of view, but more importantly, it raises hygienic concerns because they may serve as a potential reservoir of opportunistic pathogens. Although there have been several studies of pink biofilms using molecular analysis and confocal laser scanning microscopy, little is known about the spatial distributions of constituent microorganisms within pink biofilms, a crucial factor associated with the characteristics of pink biofilms. Here we show that Raman spectroscopic signatures of intracellular carotenoids and polyenes enable us to visualize pigmented microorganisms within pink biofilms in a label-free manner. We measured space-resolved Raman spectra of a pink biofilm collected from a bathroom, which clearly show resonance Raman bands of carotenoids. Multivariate analysis of the Raman hyperspectral imaging data revealed the presence of typical carotenoids and structurally similar but different polyenes, whose spatial distributions within the pink biofilm were found to be mutually exclusive. Raman measurements on individual microbial cells isolated from the pink biofilm confirmed that these distributions probed by carotenoid/polyene Raman signatures are attributable to different pigmented microorganisms. the present results suggest that Raman microspectroscopy with a focus on microbial pigments such as carotenoids is a powerful nondestructive method for studying multispecies biofilms in various environments.

Mapping of a Subgingival Dual-Species Biofilm Model Using Confocal Raman Microscopy

“…Our results also suggest that a full bacterial spectrum can be used for the differentiation of species and does not need to be limited to the selection of specific bands (Gieroba et al, 2020;Horiue et al, 2020). This allows for a more stable differentiation method, because multiple bands are considered in our method.…”

“…While they were able to determine concentrations of different chemical compounds in a specified area, the research's focus was not on the differentiation of species in a multispecies biofilms based on their chemometric spectra and lacked the statistical evaluation that resulted in the chemical maps. Horiue et al (2020) were able to map bacterial species in pink biofilms using specific pigment spectral bands and statistical methods. While it was possible to differentiate the areas, the study was unable to associate clusters to specific bacterial species.…”

Machine learning-assisted single-cell Raman fingerprinting for in situ and nondestructive classification of prokaryotes

“…The next important features center in regions 1137.5–1157.3 and 1508.8–1527.7 cm −1 . The Raman bands in these regions are well-known signatures of carotenoids ( Horiue et al., 2020 ; Withnall et al., 2003 ; Zheng et al., 2013 ), whose intensities are enhanced owing to the (pre)resonance Raman effect. In typical carotenoids, the band at ∼1152 cm −1 is assigned to the C–C stretching mode, and that at ∼1518 cm −1 the in-phase C=C stretching mode in the conjugated chain.…”

A Simple Calibration Method of Anti-Stokes–Stokes Raman Intensity Ratios Using the Water Spectrum for Intracellular Temperature Measurements

“…Raman spectra of liquid water at different temperatures were measured using a laboratory-built confocal Raman microspectrometer based on an inverted microscope, which has been described previously. 21 The output at ∼532 nm of a continuous wave semiconductor laser (Coherent, Genesis CX532-2000SLM-CDRH) was used as the Raman excitation light. The laser frequency was determined to be 18771.2 cm −1 .…”