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Fluorescence Lifetime Imaging (FLIM)

Imaging technique based on differences in the excited state decay rate

Fluorescence Lifetime Imaging (FLIM) produces an image based on the differences in the excited state decay rate from a fluorescent sample. Thus, FLIM is a fluorescence imaging technique where the contrast is based on the lifetime of individual fluorophores rather than their emission spectra. The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state by emitting a photon.
As the fluorescence lifetime does not depend on concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity it is more robust than intensity based methods. At the same time, the fluorescence lifetime depends on a wealth of environmental parameters such as pH, ion or oxygen concentration, molecular binding or the proximity of energy acceptors making it the technique of choice for functional imaging of many kinds.

Time-Correlated Single Photon Counting (TCSPC) is used to determine the fluorescence lifetime. In TCSPC, one measures the time between sample excitation by a pulsed laser and the arrival of the emitted photon at the detector. TCSPC requires a defined “start”, provided by the electronics steering the laser pulse or a photodiode, and a defined “stop” signal, realized by detection with single-photon sensitive detectors (e.g. Single Photon Avalanche Diodes, SPADs). The measurement of this time delay is repeated many times to account for the statistical nature of the fluorophores emission. The delay times are sorted into a histogram that plots the occurrence of emission over time after the excitation pulse.
In order to acquire a fluorescence lifetime image, the photons have to be attributed to the different pixels, which is done by storing the absolute arrival times of the photons additionally to the relative arrival time in respect to the laser pulse. Line and frame marker signals from the scanner of the confocal microscope are additionally recorded in order to sort the time stream of photons into the different pixels.

Consequently the essential components of a FLIM set-up are:

  • Pulsed laser source (diode lasers or multi-photon excitation)
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  • Dichroic mirror (to separate fluorescence signal from excitation light)
  • Objective (to focus the excitation light into the sample and collect fluorescence signal)
  • TCSPC unit to measure the time between excitation and fluorescence emission

Related technical and application notes:

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