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