Details
| Original language | English |
|---|---|
| Pages (from-to) | 249-260 |
| Number of pages | 12 |
| Journal | Biology and fertility of soils |
| Volume | 59 |
| Issue number | 2 |
| Early online date | 3 Feb 2023 |
| Publication status | Published - Feb 2023 |
Abstract
Soil microbial communities are involved in most biogeochemical processes creating hotspots for nutrient cycling. The spatial visualization of such soil hotspots via microscopic techniques is still challenging caused by the intrinsic fluorescence and opacity of the soil. One way to differentiate microbial cells from the heterogeneous soil matrix is a fluorescence lifetime-based technique (FLIM) with subsequent phasor plot separation; it separates and visualizes the distinctly different photon arrival times of all photons per pixel. FLIM delivers additional independent information behind intensity-based image processing and image analysis which is often hampered by, e.g., autofluorescence, resolution issues, and photobleaching artifacts caused by the prevailing minerals and organic substances. We determined characteristic fluorescence lifetime profiles of BacLight™ Green for Rhodotorula mucilaginosa and Bacillus subtilits in phosphate-buffered saline (PBS) solution and water as well as in natural, autoclaved, glucose-activated, and soil mineral particles by FLIM measurements via confocal laser scanning fluorescence microscopy. Rhodotorula mucilaginosa and Bacillus subtilits from pure cultures measured in water and PBS accounted for 1.20 (± 0.2) ns and 1.3 (± 0.1) ns respectively. The lifetime profile within the cells was rather homogeneous for both microbial species tested. This suggests stable photon arrival times for microbial strains with minor effects of matrix components as tested in PBS and water. We identified a clear difference in fluorescence lifetime profiles between microorganisms (around 1 ns) and the surrounding soil matrix (0.2 to 0.7 ns, > 3.6 ns) via phasor plot separation. The results presented raise the feasibility to extend the applicability of FLIM to other soils and their accompanying microbiota.
Keywords
- Confocal laser scanning microscopy, FLIM, Microbial separation, Phasor plot, Photon arrival times, Soil staining
ASJC Scopus subject areas
- Immunology and Microbiology(all)
- Microbiology
- Agricultural and Biological Sciences(all)
- Agronomy and Crop Science
- Agricultural and Biological Sciences(all)
- Soil Science
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In: Biology and fertility of soils, Vol. 59, No. 2, 02.2023, p. 249-260.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Using fluorescence lifetime imaging to disentangle microbes from the heterogeneous soil matrix
AU - Loeppmann, Sebastian
AU - Tegtmeier, Jan
AU - Shi, Yijie
AU - de la Fuente, Alberto Andrino
AU - Boy, Jens
AU - Guggenberger, Georg
AU - Fulterer, Andreas
AU - Fritsch, Martin
AU - Spielvogel, Sandra
N1 - Funding Information: We thank Karl-Heinz Koertje (Leica Microsystems, Mannheim) for his technical assistance. We thank Dr. Evgenia Blagodatskaya (UFZ, Halle) and Prof. Dr. Andrea Polle (Forest Botany and Tree Physiology, Georg-August University, Göttingen) for hosting preliminary microscopic experiments. We thank the Central Microscopy Facility at the Department of Biology, Kiel University and Dr. Urksa Repnik (https://www.biologie.uni-kiel.de/zm/) for technical support in using Zeiss LSM 900. Funding Information: This project was carried out in the framework of the priority program 1685 “Ecosystem Nutrition” funded by a SNF-DFG grant to Sandra Spielvogel (SS 20021E-171173).
PY - 2023/2
Y1 - 2023/2
N2 - Soil microbial communities are involved in most biogeochemical processes creating hotspots for nutrient cycling. The spatial visualization of such soil hotspots via microscopic techniques is still challenging caused by the intrinsic fluorescence and opacity of the soil. One way to differentiate microbial cells from the heterogeneous soil matrix is a fluorescence lifetime-based technique (FLIM) with subsequent phasor plot separation; it separates and visualizes the distinctly different photon arrival times of all photons per pixel. FLIM delivers additional independent information behind intensity-based image processing and image analysis which is often hampered by, e.g., autofluorescence, resolution issues, and photobleaching artifacts caused by the prevailing minerals and organic substances. We determined characteristic fluorescence lifetime profiles of BacLight™ Green for Rhodotorula mucilaginosa and Bacillus subtilits in phosphate-buffered saline (PBS) solution and water as well as in natural, autoclaved, glucose-activated, and soil mineral particles by FLIM measurements via confocal laser scanning fluorescence microscopy. Rhodotorula mucilaginosa and Bacillus subtilits from pure cultures measured in water and PBS accounted for 1.20 (± 0.2) ns and 1.3 (± 0.1) ns respectively. The lifetime profile within the cells was rather homogeneous for both microbial species tested. This suggests stable photon arrival times for microbial strains with minor effects of matrix components as tested in PBS and water. We identified a clear difference in fluorescence lifetime profiles between microorganisms (around 1 ns) and the surrounding soil matrix (0.2 to 0.7 ns, > 3.6 ns) via phasor plot separation. The results presented raise the feasibility to extend the applicability of FLIM to other soils and their accompanying microbiota.
AB - Soil microbial communities are involved in most biogeochemical processes creating hotspots for nutrient cycling. The spatial visualization of such soil hotspots via microscopic techniques is still challenging caused by the intrinsic fluorescence and opacity of the soil. One way to differentiate microbial cells from the heterogeneous soil matrix is a fluorescence lifetime-based technique (FLIM) with subsequent phasor plot separation; it separates and visualizes the distinctly different photon arrival times of all photons per pixel. FLIM delivers additional independent information behind intensity-based image processing and image analysis which is often hampered by, e.g., autofluorescence, resolution issues, and photobleaching artifacts caused by the prevailing minerals and organic substances. We determined characteristic fluorescence lifetime profiles of BacLight™ Green for Rhodotorula mucilaginosa and Bacillus subtilits in phosphate-buffered saline (PBS) solution and water as well as in natural, autoclaved, glucose-activated, and soil mineral particles by FLIM measurements via confocal laser scanning fluorescence microscopy. Rhodotorula mucilaginosa and Bacillus subtilits from pure cultures measured in water and PBS accounted for 1.20 (± 0.2) ns and 1.3 (± 0.1) ns respectively. The lifetime profile within the cells was rather homogeneous for both microbial species tested. This suggests stable photon arrival times for microbial strains with minor effects of matrix components as tested in PBS and water. We identified a clear difference in fluorescence lifetime profiles between microorganisms (around 1 ns) and the surrounding soil matrix (0.2 to 0.7 ns, > 3.6 ns) via phasor plot separation. The results presented raise the feasibility to extend the applicability of FLIM to other soils and their accompanying microbiota.
KW - Confocal laser scanning microscopy
KW - FLIM
KW - Microbial separation
KW - Phasor plot
KW - Photon arrival times
KW - Soil staining
UR - http://www.scopus.com/inward/record.url?scp=85147355842&partnerID=8YFLogxK
U2 - 10.1007/s00374-023-01704-w
DO - 10.1007/s00374-023-01704-w
M3 - Article
AN - SCOPUS:85147355842
VL - 59
SP - 249
EP - 260
JO - Biology and fertility of soils
JF - Biology and fertility of soils
SN - 0178-2762
IS - 2
ER -