Abstract
Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.
Highlights
Fluorescence lifetime imaging microscopy (FLIM) permits resesrchers mapping cell biochemistry in living cells, for instance, by detecting the state of naturally fluorescent molecules sch as NADH, or by measuring the concentration of analytes, protein-protein interactions, enzymatic activities, conformational changes of molecules, pH and viscosity with the use of fluorescent probes [1]
We have introduced a theoretical analysis to clarify and justify the need for faster single-photon counting electronics for FLIM applications
It is clear that the adoption of fast singlephoton counting electronics requires some compromise, at least with the available technologies
Summary
Fluorescence lifetime imaging microscopy (FLIM) permits resesrchers mapping cell biochemistry in living cells, for instance, by detecting the state of naturally fluorescent (often meatbolic related) molecules sch as NADH, or by measuring the concentration of analytes, protein-protein interactions, enzymatic activities, conformational changes of molecules, pH and viscosity with the use of fluorescent probes [1]. Typical wide-field FLIM microscopes required only a few seconds of exposure to excitation light per image, during which, intensified gated or modulated cameras temporally slice-through exponentially decaying (time-domain detection [2], figure 1(a)) or sinusoidally modulated fluorescence signals (frequency-domain detection [3], figure 1(b)). Even when operated in such single-shot modality, intensified cameras lose a significant amount of light by gating photons off during acquisition (figures 1(a), (c)). Often, these losses results in lower precision than techniques implemented with laser scanning microscopes. Laser scanning microscopes can provide higher resolution than conventional wide-field microscopes with the additional advantage that electronics and optical arrangements for single-pixel detection can grow in complexity compared to two-dimensional imagers (e.g., fast and precise digitizers and hyperspectral detection)
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