Everything about Fluorescence Lifetime Imaging totally explained
Fluorescence lifetime imaging or
FLIM is a powerful tool for producing an image based on the differences in the exponential decay rate of the
fluorescence from a fluorescent sample. It can be used as an imaging technique in
confocal microscopy and other microscope systems.
The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample. FLIM is very useful for biomedical tissue imaging, allowing to probe greater tissue depths than conventional fluorescence microscopy.
Fluorescence lifetimes
A
fluorophore which is
excited by a
photon will drop to the
ground state with a certain probability based on the decay rates through a number of different (radiative and/or nonradiative) decay pathways. To observe fluorescence, one of these pathways must be by
spontaneous emission of a photon. In the
ensemble description, the fluorescence emitted will decay with time according to
»
The decay function (and corresponding lifetimes) can't be recovered by direct
deconvolution using
Fourier transforms because division by zero will produce errors and noise will be amplified. However, the instrumental response of the source, detector, and electronics can be measured, usually from scattered excitation light. The IRF can then be convolved with a trial decay function to produce a calculated fluorescence, which can be compared to the measured fluorescence. The parameters for the trial decay function can be varied until the calculated and measured fluorescence curves fit well. This process is known as reconvolution or reiterative convolution, and can be performed quickly by several software packages.
Phase modulation
Alternatively, fluorescence lifetimes can be determined in the frequency domain by a phase-modulated method. The intensity of a
continuous wave source is modulated at high frequencey, by an
acousto-optic modulator for example, which will modulate the fluorescence. Since the excited state has a lifetime, the fluorescence will be delayed with respect to the excitation signal, and the lifetime can be determined from the phase shift. Also, y-components to the excitation and fluorescence sine waves will be modulated, and lifetime can be determined from the modulation ratio of these y-components. Hence, 2 values for the lifetime can be determined from the phase-modulation method.
Further Information
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