Time-resolved fluorescence microscopy for measuring specific coenzymes in methanogenic bacteria

Schneckenburger, Herbert; Reuter, Bernd; Schoberth, Siegfried M.

doi:10.1016/s0003-2670(00)81514-7

Cited by 10 publications

(3 citation statements)

References 8 publications

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“…This may also apply to cell drying and two glycerol-based methods [ 47 ] which severely altered both cell integrity (analyzed by abundance reduction of F 420 autofluorescent cells; − 66.8, − 46.8, − 76%), and fluorescence intensity (analyzed by mean FI reduction; − 78.1, − 7, − 38.2%) and, consequently, are also not recommended. Photobleaching can diminish the cofactor F 420 autofluorescence intensity [ 14 , 48 , 49 ] and potentially impact microscopic counting. However, flow cytometry analyses only require exposure times of 0.75–1.5 µs per cell and will therefore not bias the quantification of methanogenic archaea.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

et al. 2017

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BackgroundThe widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious.ResultsIn this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL−1 and 1.8 x 109 (± 1.1 × 108) cells mL−1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results.ConclusionsThe presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0793-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

et al. 2017

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“…In experiments by Schneckenburger et al, it was discovered that Methanobacterium (which uses F 420 ) exhibited different photobleaching behaviors based on whether the methanobacteria were active or inactive [10]. The active methanobacteria photobleached at a slower rate than that of inactive methanobacteria, thus authors were able to distinguish between active and inactive organisms by measuring their respective photobleaching rates.…”

Section: Introductionmentioning

confidence: 99%

Differentiating between live and dead Mycobacterium smegmatis using autofluorescence

Wong

Pawłowski

et al. 2016

Tuberculosis

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While there have been research efforts to find faster and more efficient diagnostic techniques for tuberculosis (TB), it is equally important to monitor a patient’s response to treatment over time, especially with the increasing prevalence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB. Between sputum smear microscopy, culture, and GeneXpert, only culture can verify viability of mycobacteria. However, it may take up to six weeks to grow Mycobacterium tuberculosis (Mtb), during which time the patient may have responded to treatment or the mycobacteria are still viable because the patient has MDR or XDR TB. In both situations, treatment incurs increased patient costs and makes them more susceptible to host-drug effects such as liver damage. Coenzyme Factor 420 (F420) is a fluorescent coenzyme found naturally in mycobacteria, with an excitation peak around 420 nm and an emission peak around 470 nm. Using Mycobacterium smegmatis, we show that live and dead mycobacteria undergo different rates of photobleaching over a period of 2 min. These preliminary experiments suggest that the different photobleaching rates could be used to help monitor a patient’s response to TB treatment. In future studies, we propose to describe these experiments with Mtb as both M. smegmatis and Mtb use F420.

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“…[30][31][32] Advances in high-repetition lasers for pulse excitation, TCSPC, and frequency-domain methods have permitted more reliable fluorescence lifetimes to be obtained from microscopically portioned sections. Several groups have developed TRFM using argon ionpulsed lasers, 33,34 modelocked picosecond dye lasers, [35][36][37][38] and the TCSPC technique. The frequency-domain technique also has been utilized for fluorescence microscopy.…”

Section: Introductionmentioning

confidence: 99%

[30] Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier

Szmacinski

Lakowicz²,

Johnson³

1994

Methods in Enzymology

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In the previous sections we demonstrated imaging of intracellular Ca2+ using our approach to FLIM. What other analytes can be imaged using FLIM? We have now characterized the lifetime of a good number of ion indicators. Based on these studies we know that Cl- can be imaged using FLIM with probes such as SPQ or MQAE, pH can be imaged using resorufin and probes of the SNAFL and SNARF (Molecular Probes) series, and Mg2+ can be imaged using Magnesium Green, Mag-quin-2, or Mag-quin-1 (Molecular Probes). At present, the probe for K+, as PBFI, are just adequate as a lifetime probe, but it seems likely that newer probes for Na+ (Sodium Green) and K+ will be practical for effective imaging. Of course, imaging of oxygen is possible using a wide variety of fluorophores. It should be noted that a wide variety of substances and/or phenomena are known to alter decay times, acting as quenchers. These include the phenomena of resonance energy transfer, collisional quenching, temperature effects, and viscosity effects. Also, the FLIM method is not limited to microscopic objects but can be possibly used in remote imaging of any object. Hence, FLIM will allow the imaging of the chemical and physical properties of objects based on the effects of the local environment on the decay kinetics of fluorophores. The instrumentation for FLIM is presently complex and requires a moderately complex laser source, a gain-modulated image intensifier, and a slow-scan CCD camera. However, one can readily imagine the instrumentation becoming rather compact, and even all solid-state, owing to advances in laser and CCD technologies and, more importantly, advances in probe chemistry. To be specific, the dye laser shown in Fig. 1 may be replaced by a simpler UV laser, such as the 354 nm HeCd laser which has become available (Fig. 11). Intensity modulation of a continuous wave sources can be accomplished with acoustooptic modulators. The scientific slow-scan CCD cameras are presently rather expensive, but they are used in the present instrumentation because of their linearity and high dynamic range. However, the increasing use of CCD detectors suggest that even the scientific-grade CCD cameras will soon become less costly. Additionally, the frame rates of these detectors continue to increase in response to the need for faster imaging. Furthermore, the performance of the video CCD cameras is increasing, as seen by the introduction of 10-bit video analog-to-digital (A/D) converters.(ABSTRACT TRUNCATED AT 400 WORDS)

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Time-resolved fluorescence microscopy for measuring specific coenzymes in methanogenic bacteria

Cited by 10 publications

References 8 publications

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

Differentiating between live and dead Mycobacterium smegmatis using autofluorescence

[30] Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier

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