The fluorescence lifetime (FLT) of the fluorophore, rather than its intensity, is used to create the image in FLIM.
Fluorescence lifetime depends on the local micro-environment of the fluorophore, thus precluding any erroneous measurements in fluorescence intensity due to change in brightness of the light source, background light intensity or limited photo-bleaching.
This technique also has the advantage of minimizing the effect of photon scattering in thick layers of sample.
Being dependent on the micro-environment, lifetime measurements have been used as an indicator for pH,[1] viscosity[2] and chemical species concentration.
When a population of fluorophores is excited by an ultrashort or delta pulse of light, the time-resolved fluorescence will decay exponentially as described above.
However, if the excitation pulse or detection response is wide, the measured fluorescence, d(t), will not be purely exponential.
The instrumental response of the source, detector, and electronics can be measured, usually from scattered excitation light.
Recovering the decay function (and corresponding lifetimes) poses additional challenges as division in the frequency domain tends to produce high noise when the denominator is close to zero.
Time-correlated single-photon counting (TCSPC) is usually employed because it compensates for variations in source intensity and single photon pulse amplitudes.
Using commercial TCSPC equipment a fluorescence decay curve can be recorded with a time resolution down to 405 fs.
More specifically, TCSPC records times at which individual photons are detected by a fast single-photon detector (typically a photo-multiplier tube (PMT) or a single photon avalanche photo diode (SPAD)) with respect to the excitation laser pulse.
Multi-channel PMT systems with 16[7] to 64 elements have been commercially available, whereas the recently demonstrated CMOS single-photon avalanche diode (SPAD)-TCSPC FLIM systems can offer even higher number of detection channels and additional low-cost options.
Before the pulse reaches the sample, some of the light is reflected by a dichroic mirror and gets detected by a photodiode that activates a delay generator controlling a gated optical intensifier (GOI) that sits in front of the CCD detector.
[9][10] In recent years integrated intensified CCD cameras entered the market.
These cameras consist of an image intensifier, CCD sensor and an integrated delay generator.
ICCD cameras with shortest gating times of down to 200ps and delay steps of 10ps allow sub-nanosecond resolution FLIM.
An advantage of PMT-based or camera-based frequency domain FLIM is its fast lifetime image acquisition making it suitable for applications such as live cell research.
The most widely used technique is the least square iterative re-convolution which is based on the minimization of the weighted sum of the residuals.
In this technique theoretical exponential decay curves are convoluted with the instrument response function, which is measured separately, and the best fit is found by iterative calculation of the residuals for different inputs until a minimum is found.
Besides experimental difficulties, including the wavelength dependent instrument response function, mathematical treatment of the iterative de-convolution problem is not straightforward and it is a slow process which in the early days of FLIM made it impractical for a pixel-by-pixel analysis.
One of the major and straightforward techniques in this category is the rapid lifetime determination (RLD) method.
RLD calculates the lifetimes and their amplitudes directly by dividing the decay curve into two parts of equal width
One major drawback of this method is that it cannot take into account the instrument response effect and for this reason the early part of the measured decay curves should be ignored in the analyses.
These approaches are faster than the deconvolution based methods but they suffer from truncation and sampling problems.
FLIM has primarily been used in biology as a method to detect photosensitizers in cells and tumors as well as FRET in instances where ratiometric imaging is difficult.
[17] Time domain FLIM (tdFLIM) has also been used to show the interaction of both types of nuclear intermediate filament proteins lamins A and B1 in distinct homopolymers at the nuclear envelope, which further interact with each other in higher order structures.
[19] In neurons, FLIM imaging using pulsed illumination has been used to study Ras,[20] CaMKII, Rac, and Ran[21] family proteins.
FLIM has been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetic compounds.
[22] Multi-photon FLIM is increasingly used to detect auto-fluorescence from coenzymes as markers for changes in mammalian metabolism.
[23][24] Since the fluorescence lifetime of a fluorophore depends on both radiative (i.e. fluorescence) and non-radiative (i.e. quenching, FRET) processes, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor.