Using fluorescence lifetime imaging to disentangle microbes from the heterogeneous soil matrix

Soil microbial communities drive most biogeochemical processes and create hotspots of nutrient cycling. However, spatial visualization of microorganisms in these soil hotspots at the microscopic scale remains challenging due to the intrinsic fluorescence and opacity of soil matrices. One promising approach to distinguish microbial cells from the heterogeneous soil background is fluorescence lifetime imaging microscopy (FLIM) combined with phasor plot analysis. This technique separates and visualizes distinct photon arrival times on a per-pixel basis, providing information independent of fluorescence intensity. As a result, FLIM overcomes limitations of intensity-based imaging caused by autofluorescence, limited resolution, and photobleaching artifacts associated with minerals and organic matter.In this study, we determined characteristic fluorescence lifetime profiles of BacLight™ Green–stained Rhodotorula mucilaginosa and Bacillus subtilis using FLIM via confocal laser scanning fluorescence microscopy. Measurements were conducted in phosphate-buffered saline solution (PBS), water, and in natural, autoclaved, glucose-activated soils, as well as soil mineral particles. In pure cultures, fluorescence lifetimes were 1.20 ± 0.2 ns for R. mucilaginosa and 1.30 ± 0.1 ns for B. subtilis in both water and PBS. Fluorescence lifetimes within individual cells were spatially homogeneous for both species, indicating stable photon arrival times and only minor matrix effects under the tested conditions.Using phasor plot analysis, we observed a clear separation between microbial fluorescence lifetimes (approximately 1 ns) and those of the surrounding soil matrix (0.2–0.7 ns and > 3.6 ns). These findings demonstrate the feasibility of using FLIM to discriminate microbial cells from complex soil backgrounds and suggest strong potential for extending this approach to other soil types and their associated microbiota.