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

Loeppmann, Sebastian; Tegtmeier, Jan; Shi, Yuanming; Fuente, Alberto Andrino de la; Boy, Jens; Guggenberger, Georg; Fulterer, Andreas; Fritsch, Martin; Spielvogel, Sandra

doi:10.1007/s00374-023-01704-w

Cited by 3 publications

(3 citation statements)

References 51 publications

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“…A 2023 study used FLIM to see labelled bacteria in soils. 50 Detection of unpigmented bacteria by native autofluorescence remains difficult. In pure cultures, laser power and acquisition times may be increased in order to observe native fluorescence of unpigmented organisms, which largely results from NAD(P)H and flavins.…”

Section: Discussionmentioning

confidence: 99%

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The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

Chmykh,

Nadeau

2024

Journal of Microscopy

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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.LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high‐power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.

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Section: Discussionmentioning

confidence: 99%

“…The authors reported that signals from fungi were much stronger than those from unlabelled bacteria, though unlabelled bacteria could sometimes be seen. A 2023 study used FLIM to see labelled bacteria in soils 50 . Detection of unpigmented bacteria by native autofluorescence remains difficult.…”

Section: Discussionmentioning

confidence: 99%

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

Chmykh,

Nadeau

2024

Journal of Microscopy

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“…Upon image acquisition, the distinctly different photon arrival times of all photons per pixel are separated in a procedure called phasor plot separation, and fluorescence decay can be visualized. In the first study, clear differences were seen in fluorescence lifetime profiles between the fluorescence signals of microorganisms and the soil matrix, thus demonstrating the applicability of the methods for soil samples (Loeppmann et al., 2023). Potential limitations of this approach are slow image acquisition and long processing times.…”

Section: Microbial Biogeography In and At Microaggregatesmentioning

confidence: 91%

Architecture of soil microaggregates: Advanced methodologies to explore properties and functions

Amelung,

Tang,

Siebers

et al. 2023

J. Plant Nutr. Soil Sci.

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The functions of soils are intimately linked to their three‐dimensional pore space and the associated biogeochemical interfaces, mirrored in the complex structure that developed during pedogenesis. Under stress overload, soil disintegrates into smaller compound structures, conventionally named aggregates. Microaggregates (<250 µm) are recognized as the most stable soil structural units. They are built of mineral, organic, and biotic materials, provide habitats for a vast diversity of microorganisms, and are closely involved in the cycling of matter and energy. However, exploring the architecture of soil microaggregates and their linkage to soil functions remains a challenging but demanding scientific endeavor. With the advent of complementary spectromicroscopic and tomographic techniques, we can now assess and visualize the size, composition, and porosity of microaggregates and the spatial arrangement of their interior building units. Their combinations with advanced experimental pedology, multi‐isotope labeling experiments, and computational approaches pave the way to investigate microaggregate turnover and stability, explore their role in element cycling, and unravel the intricate linkage between structure and function. However, spectromicroscopic techniques operate at different scales and resolutions, and have specific requirements for sample preparation and microaggregate isolation; hence, special attention must be paid to both the separation of microaggregates in a reproducible manner and the synopsis of the geography of information that originates from the diverse complementary instrumental techniques. The latter calls for further development of strategies for synlocation and synscaling beyond the present state of correlative analysis. Here, we present examples of recent scientific progress and review both options and challenges of the joint application of cutting‐edge techniques to achieve a sophisticated picture of the properties and functions of soil microaggregates.

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Soil zymography: A decade of rapid development in microbial hotspot imaging

Bilyera,

Kuzyakov

2024

Soil Biology and Biochemistry

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

Cited by 3 publications

References 51 publications

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates

Architecture of soil microaggregates: Advanced methodologies to explore properties and functions

Soil zymography: A decade of rapid development in microbial hotspot imaging

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