Fast Timing Techniques in FLIM Applications

Abstract

Fluorescence lifetime imaging (FLIM) is increasingly used in many scientific disciplines, including biological and medical research, materials science and chemistry. The fluorescence label is not only used to indicate its location, but also to probe its immediate environment, via its fluorescence lifetime. This allows FLIM to monitor and image the cellular microenvironment including the interaction between proteins in their natural environment. It does so with high specificity and sensitivity in a non-destructive and minimally invasive manner, providing both structural and functional information. Time-Correlated Single Photon Counting (TCSPC) is a popular, widely used, robust and mature method to perform FLIM measurements. It is a sensitive, accurate and precise method of measuring photon arrival times after an excitation pulse, with the arrival times not affected by photobleaching, excitation or fluorescence intensity fluctuations. It has a very large dynamic range, and only needs a low illumination intensity. Different methods have been developed to advance fast and accurate timing of photon arrival. In this review a brief history of the development of these methods is given, and their merits are discussed in the context of their applications in FLIM.

FLIM as a Promising Tool for Cancer Diagnosis and Treatment Monitoring

“…2 b. In FLIM, single-photon detectors and photon timing techniques are incorporated into this setup for lifetime measurement [ 16 ]. Single-photon detectors in FLIM are typically based on photon amplification through electrical current generation when photons impact a photovoltaic such as the photomultiplier tube (PMT) or avalanche photodiode (APD).…”

Superconducting nanowire single-photon sensing of cerebral blood flow

“…SNSPDs are used in optical quantum information, telecommunication, and space communication. 15 SNSPDs have also recently been used in fluorescence lifetime imaging microscopy 16 but never before in biomedical applications. To our knowledge, this is the first application of SNSPDs in humans to improve DCS performance.…”

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

“…The need to reach higher acquisition speeds may require a trade-off in other instrument specifications such as the number of bins used to sample fluorescence decays or the instrument response function (IRF), i.e. the uncertainty over the sampled arrival time of each photon caused by variabilities in the excitation pulse, the transit time spread of the detector, and the time-jitter of the detector and electronics [ 16 ]. The signal measured on a system, M(t) , is thus represented by the convolution integral of the fluorescence decay, D(t) , and the instrument response function, IRF(t) [ 1 , 2 , 17 , 18 ].…”