Fast single-cell biochemistry: theory, open source microscopy and applications

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.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…[88][89][90][91][92][93][94] A detailed discussion on challenges and current approaches to improve FLIM can be found in a previous review. 95 Other time-domain methods include time-gating (TG) and pulse sampling. In TG FLIM, following a short excitation pulse, the fluorescence decay is directly sampled at two or more time gates that are sequentially delayed from the excitation pulse.…”

The biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics

“…the inability of resolving a second photon while the detection system is still busy handling the electrical trace of the first (due to the combined effect of the detector and the electronics deadtimes), or difficulties in detecting multiple photons per light pulse. We have made significant progress in ameliorating pulse pile-up effects, for example, by utilizing multi-hit time-to-digital [7,8] or multiplexed time-to-analog converters [9], ultra-fast digitizers [10], fast detectors (e.g. hybrid photomultipliers [11]), and detector arrays [12][13][14][15].…”

How many photons are needed for FRET imaging?

“…Although the analysis we provided is representative of the limits on precision imposed by photon-statistics for lifetime determination and thus FRET/FLIM, there are also many other implementations of FLIM that can be successfully used for FRET estimation [1,5,24]. At high count-rates, for instance, TCSPC deteriorates its precision and accuracy because of photon-losses and distortions of the experimental decays caused by pulse pile-up and detector dead-time [12,56,57]. These losses were not accounted in this framework.…”