Time-gated fluorescence microscopy in cellular and molecular biology.
An experimental set-up for time-gated fluorescence spectroscopy and microscopy is described, and some recent applications in cellular and molecular biology are summarized. Selective detection of intrinsic fluorophores, in particular nicotinamide adenine dinucleotide (NADH) and flavins was demonstrated in living cells. Non-radiative energy transfer from reduced NADH to the mitochondrial marker rhodamine 123 was evaluated for probing mitochondrial malfunction in living cells. An increase of "energy transfer efficacy" up to a factor 4 was detected after inhibition of enzyme complexes of the respiratory chain. Two different fluorescence lifetimes of calcium orange were evaluated, whose relative intensities depended on calcium concentration. Therefore, fluorescence measured within two different time gates appeared to be suitable for ratio fluorometry of calcium. Time-gated fluorescence spectra of the membrane marker laurdan showed more pronounced changes than steady state spectra when temperature was increased from 24 degrees C to 38 degrees C. This may improve measurements of intracellular temperature. Time-gated detection of small amounts of porphyrins and their discrimination from a large fluorescent background caused by chlorophyll in transgenic tobacco plants again proved the advantages of time-gated fluorescence spectroscopy.
Institut für Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm, Germany.
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Schneckenburger
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Gschwend
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Sailer
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Mock
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Strauss
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000032:analysis; D002734:Chlorophyll / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis*; D007091:Image Processing, Computer-Assisted; D007848:Laurates / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D009930:Organic Chemicals; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D012441:Saccharomyces cerevisiae / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014026:Nicotiana / Q000235:genetics
D005456:Fluorescent Dyes; D007848:Laurates; D009930:Organic Chemicals; C071335:calcium orange; D002734:Chlorophyll; D015081:2-Naphthylamine; C065580:laurdan