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 nondestructive 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 or 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.
Highlights
In 1961, Lowell Bollinger and George Thomas generalized the scintillation measurements to include any type of radiation [14], and flashlamps with optical pulse widths of around 2 ns became available in the 1960s, enabling TimeCorrelated Single Photon Counting (TCSPC): This is essentially a delayed coincidence method, whereby the arrival time of a single photon is measured relative to an excitation pulse, and this can be done with a few picosecond precision [15]
Besides scanning with a point-detector to form an image of the sample, microchannel plate (MCP)-based detectors and more recently single photon avalanche diode (SPAD) arrays can be used for wide-field TCSPC Fluorescence lifetime imaging (FLIM) [47]
The development of both single point and widefield single photon detection methods has continued over the decades, and while the old technologies continue to be used and improved, there have been new developments that show great promise, including SPAD arrays and detectors based on superconducting detector technology
Summary
The quest for precise timing of light signals can be traced back to 1638, when Galileo Galilei (15641642), performed an experiment using two observers with lanterns and manual shutters stationed on two well-separated hilltops He suspected that light did not travel instantaneously, this kind of terrestrial approach was far too slow to observe the speed of light experimentally. Astronomical measurements provided a way forward: In 1675, the Danish astronomer Ole Rømer (1644–1710) observed the eclipses of the innermost moon of Jupiter, Io, at different times of the year and proposed a finite speed of light to account for timing differences of the moon emerging from the shadow of Jupiter, as seen from earth In another astronomical approach around 50 years later, in 1728, the British astronomer James Bradley (1693–1762) calculated the speed of light from stellar aberration [1]. At King’s College London, Charles Wheatstone (1802–1875) experimented with rotating mirrors to measure the duration of electric sparks [3]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
