Sponsor
This research was supported by a contract from Marie-Veronique Inc. to Portland State University and by NASA ICEE-2 Europa Luminescence Microscope (ELM) (80NSSC19M0122). We acknowledge expert technical assistance by staff in the Advanced Light Microscopy Core in the Department of Neurology and Jungers Center at Oregon Health and Science University. We thank Wolfgang Becker for helpful discussions.
Published In
Journal of Microscopy
Document Type
Article
Publication Date
1-2024
Subjects
Fluorescence, Fluorescence spectrometry -- Research
Abstract
The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology.
Rights
Copyright (c) 2024 The Authors
This work is licensed under a Creative Commons Attribution 4.0 International License.
Locate the Document
DOI
10.1111/jmi.13264
Persistent Identifier
https://archives.pdx.edu/ds/psu/41093
Citation Details
Chmykh, Y., & Nadeau, J. L. (2024). The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates. Journal of Microscopy.