TCSPC Lifetime.

The fluorescence lifetime is a measure of how long a photon exicted electron will remain in a molecules excited state before returning to the ground state via the emission of a lower energy (longer wavelength) photon. When combined with an imaging microscope system it is possible to measure the fluorescence decay of a molecular species at every point in the optical field of view. This method is known as Fluorescence Lifetime Imaging Microscopy (FLIM).

The two most commonly used general techniques to acquire FLIM data are frequency domain and time domain. These can be divided into sub-groups depending upon the technology and/or the method of analysis used. The first general method, frequency domain FLIM (fd-FLIM), is covered in another Technique section (Frequency Domain), leaving that of time domain FLIM (td-FLIM) to be covered here.

 td-FLIM can be performed one of two ways. Either the fluorescence decay is recorded for an entire field of view by use of a gated CCD camera (known as wide-field td-FLIM) or as in a confocal or multi-photon microscope the decay is recorded for each pixel in a scanning manner in a technique known as Time Correlated Single Photon Counting (TCSPC) FLIM. Both ways have their advantages and disadvantages and must be weighed up considering your experiments and what you want to get from it.

The first consideration would be acquisition time. What determines this is the rate at which your biology occurs at is it a rapid release of calcium on the scale of a few seconds? or is it a fairly static longer term process over the course of a few minutes? For fast occurring processes, unless your sample is extremely bright (not always possible while remaining physiological and having healthy cells) your choice would be wide-field FLIM as the whole filed of view can be imaged on the seconds scale rather than the minutes scale of TCSPC FLIM. Each td-FLIM method groups the arriving photons on the detector into time bins some time after the laser pulse has generated the fluorescence emission as shown on the figure below. Another factor which allows the wide-field FLIM to be faster is that typically less than 10 bins are used as compared to the 64 bins used for TCSPC measurements.

Description of techniques:

The ultra-fast pulsed laser generates a train of light pulses which excites the fluorophores in the sample.  The generated fluorescence is then collected and imaged on to the GOI which is a Gated Optical Intensifier, a photocathode which converts the incoming photons into electrons which then travel a short distance and generates secondary photons which are detected by the CCD camera. The GOI essentially acts as a very controlled and fast shutter allowing the camera to detect the light from the sample at specific times relative to the pulse from the laser.

From this, a decay curve is assembled to which an exponential curve is fit, with the decay constants corresponding to the fluorescence decay time of the sample.

 

TCSPC FLIM detection works in a similar manner in that it uses a pulsed laser system to generate the fluorescence from your sample. The laser pulse is detected by a photodetector which starts a timing circuit (TAC). This clock continues until it is stopped by a signal from the photodetector in the fluorescence emission path. A histogram is built up of many such events, fitting an exponential to the histogram leads to the fluorescence decay time. The limitation of only detecting a single photon per excitation event increases the time it takes to collect enough emission to be able to fit the decay successfully.

Information courtesy of Dr Ewan McGee In vivo Imaging specialist BAIR facility.

 

 

 

 

 

 

 

 

 

 

 

 

 


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Last modified: 02/25/16