Abstract
In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.
Highlights
The precise regulation of intracellular homeostasis is the basis of all cellular functions, as small failures can potentially lead to severe dysfunctions
Fluorescence lifetime imaging microscopy (FLIM) uses the fluorescence decay time as the recording parameter, which can be directly linked to changes in the respective physiological parameter
A recently developed time-correlated single-photon counting (TCSPC) board TimeHarp 260 PICO was evaluated for FLIM/PLIM measurements[14]
Summary
The precise regulation of intracellular homeostasis is the basis of all cellular functions, as small failures can potentially lead to severe dysfunctions. It must be emphasized that the term ‘multiparameter detection’ in this context means the simultaneous observation of at least two physiological parameters by using the appropriate number of analyte-sensitive fluorophores and detecting changes in their fluorescent properties In this case, each fluorescent sensor can be detected using spectral separation via different excitation or emission wavelengths. Reports on the solely time-resolved separation of different fluorescent sensors are limited[9,10] One reason for this is the number of photons required for a reliable analysis of multiexponential fluorescence decay behaviour, which increases dramatically as the number of decay time components increases, leading to longer acquisition times and possible cell damage[11]. A pattern-matching approach could be applied as an alternative[13] Another approach is the combination of fluorescence and phosphorescence lifetime imaging (FLIM/ PLIM), since it provides access to the discrimination of luminescent sensors with notably separated luminescence decay times. The multi-stop functionality and short dead time of the board allows several phosphorescence photons to be captured in a single excitation cycle, which is crucial to limit the necessary acquisition time
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