Fluorescence lifetime imaging
Encyclopedia
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique 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
, two-photon excitation microscopy
, and multiphoton tomography.
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.
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
where
.
In the above,
is time, 
is the fluorescence lifetime, 
is the initial fluorescence at 
, and 
are the rates for each decay pathway, at least one of which must be the fluorescence decay rate 
. More importantly, the lifetime, 
, is independent of the initial intensity of the emitted light. This can be utilized for making non-intensity based measurements in chemical sensing.
, which allows one to view contrast between materials with different fluorescence decay rates (even if those materials fluoresce at exactly the same wavelength), and also produces images which show changes in other decay pathways, such as in FRET imaging
.
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, M(t), will not be purely exponential. The instrumental response function, IRF(t) will be convolved
or blended with the decay function, F(t).
The decay function (and corresponding lifetimes) cannot be recovered by direct deconvolution
using Fourier transform
s 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.
(useful in determining goodness of fit
during reconvolution). More specifically, TCSPC records times at which individual photons are detected by something like a photo-multiplier tube (PMT) or an avalanche photo diode (APD) after a single pulse. The recordings are repeated for additional pulses and after enough recorded events, one is able to build a histogram of the number of events across all of these recorded time points. This histogram can then be fit to an exponential function that contains the exponential lifetime decay function of interest, and the lifetime parameter can accordingly be extracted.
source is modulated at high frequency, 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. Consequently, if the lifetimes that are extracted from the y-component and the phase do not match, it means that you have more than one lifetime species in your sample.
, CaMKII, Rac
, and Ran family proteins. FLIM has been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetical compounds.
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
from a fluorescent sample. It can be used as an imaging technique in confocal microscopy
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
, two-photon excitation microscopy
Two-photon excitation microscopy
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite...
, and multiphoton tomography.
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.
Fluorescence lifetimes
A fluorophoreFluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
which is excited
Excited state
Excitation is an elevation in energy level above an arbitrary baseline energy state. In physics there is a specific technical definition for energy level which is often associated with an atom being excited to an excited state....
by a photon
Photon
In physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...
will drop to the ground state
Ground state
The ground state of a quantum mechanical system is its lowest-energy state; the energy of the ground state is known as the zero-point energy of the system. An excited state is any state with energy greater than 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
Spontaneous emission
Spontaneous emission is the process by which a light source such as an atom, molecule, nanocrystal or nucleus in an excited state undergoes a transition to a state with a lower energy, e.g., the ground state and emits a photon...
of a photon. In the ensemble description, the fluorescence emitted will decay with time according to
where
.In the above,
is time, is the fluorescence lifetime, is the initial fluorescence at , and are the rates for each decay pathway, at least one of which must be the fluorescence decay rate . More importantly, the lifetime, , is independent of the initial intensity of the emitted light. This can be utilized for making non-intensity based measurements in chemical sensing.Measurement and processing
Fluorescence-lifetime imaging yields images with the intensity of each pixel determined by, which allows one to view contrast between materials with different fluorescence decay rates (even if those materials fluoresce at exactly the same wavelength), and also produces images which show changes in other decay pathways, such as in FRET imagingFluorescence resonance energy transfer
Förster resonance energy transfer , also known as fluorescence resonance energy transfer, resonance energy transfer or electronic energy transfer , is a mechanism describing energy transfer between two chromophores.A donor chromophore, initially in its electronic excited state, may transfer energy...
.
Pulsed illumination
Fluorescence lifetimes can be determined in the time domain by using a pulsed source.When a population of fluorophores is excited by an ultrashort or delta
Dirac delta function
The Dirac delta function, or δ function, is a generalized function depending on a real parameter such that it is zero for all values of the parameter except when the parameter is zero, and its integral over the parameter from −∞ to ∞ is equal to one. It was introduced by theoretical...
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, M(t), will not be purely exponential. The instrumental response function, IRF(t) will be convolved
Convolution
In mathematics and, in particular, functional analysis, convolution is a mathematical operation on two functions f and g, producing a third function that is typically viewed as a modified version of one of the original functions. Convolution is similar to cross-correlation...
or blended with the decay function, F(t).
The decay function (and corresponding lifetimes) cannot be recovered by direct deconvolution
Deconvolution
In mathematics, deconvolution is an algorithm-based process used to reverse the effects of convolution on recorded data. The concept of deconvolution is widely used in the techniques of signal processing and image processing...
using Fourier transform
Fourier transform
In mathematics, Fourier analysis is a subject area which grew from the study of Fourier series. The subject began with the study of the way general functions may be represented by sums of simpler trigonometric functions...
s 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.
TCSPC
Time-correlated single-photon counting (TCSPC) is usually employed because variations in source intensity and photoelectron amplitudes are ignored, the time resolution can be upwards of 4 ps, and the data obeys Poisson statisticsPoisson distribution
In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time and/or space if these events occur with a known average rate and independently of the time since...
(useful in determining goodness of fit
Goodness of fit
The goodness of fit of a statistical model describes how well it fits a set of observations. Measures of goodness of fit typically summarize the discrepancy between observed values and the values expected under the model in question. Such measures can be used in statistical hypothesis testing, e.g...
during reconvolution). More specifically, TCSPC records times at which individual photons are detected by something like a photo-multiplier tube (PMT) or an avalanche photo diode (APD) after a single pulse. The recordings are repeated for additional pulses and after enough recorded events, one is able to build a histogram of the number of events across all of these recorded time points. This histogram can then be fit to an exponential function that contains the exponential lifetime decay function of interest, and the lifetime parameter can accordingly be extracted.
Gating method
Pulse excitation is still used in this method. Before the pulse reaches the sample though, 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 your CCD detector. The GOI only allows for detection for the fraction of time when it is open after the delay. Thus, with an adjustable delay generator, one is able to collect fluorescence emission after multiple delay times encompassing the time range of the fluorescence decay of the sample.Phase modulation
Alternatively, fluorescence lifetimes can be determined in the frequency domain by a phase-modulated method. The intensity of a continuous waveContinuous wave
A continuous wave or continuous waveform is an electromagnetic wave of constant amplitude and frequency; and in mathematical analysis, of infinite duration. Continuous wave is also the name given to an early method of radio transmission, in which a carrier wave is switched on and off...
source is modulated at high frequency, by an acousto-optic modulator
Acousto-optic modulator
An acousto-optic modulator , also called a Bragg cell, uses the acousto-optic effect to diffract and shift the frequency of light using sound waves . They are used in lasers for Q-switching, telecommunications for signal modulation, and in spectroscopy for frequency control. A piezoelectric...
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. Consequently, if the lifetimes that are extracted from the y-component and the phase do not match, it means that you have more than one lifetime species in your sample.
Applications
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. The technique was developed in the late 1980s and early 1990s (Bugiel et al. 1989. König 1989, before being more widely applied in the late 1990s. In cell culture, it has been used to study EGF receptor signaling and ErbB1 receptor trafficking. FLIM imaging is particularly useful in neurons, where light scattering by brain tissue is problematic for ratiometric imaging. In neurons, FLIM imaging using pulsed illumination has been used to study RasRas
Ras is the name given to a family of related proteins found inside cells, including human cells. All Ras protein family members belong to a class of protein called small GTPase, and are involved in transmitting signals within cells...
, CaMKII, Rac
RAC
-Companies:* Rent-A-Center, an American public furniture and electronics rent to own company* Royal Automobile Club, a private club in Pall Mall, London* RAC plc, a breakdown company in the United Kingdom...
, and Ran family proteins. FLIM has been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetical compounds.