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Within the scientific study of a molecule's
color Color (American English) or colour (British English) is the visual perceptual property deriving from the spectrum of light interacting with the photoreceptor cells of the eyes. Color categories and physical specifications of color are assoc ...
, Fluorescence intensity decay shape microscopy (FIDSAM) is a
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of mi ...
technique, which utilizes the time evolution of fluorescence emission after a pulsed excitation to analyse the decay statistics of an excited
chromophore A chromophore is the part of a molecule responsible for its color. The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
. The main application of FIDSAM is the discrimination of unspecific autofluorescent background signal from the target signal of a dedicated chromophore.


Principle

The FIDSAM method analyses the number of different molecules contributing to a measured fluorescence signal. Assuming a pure fluorescent dye solution in an isotropic surrounding, the individual emitters are indistinguishable. Accordingly, they obey the same fluorescence emission statistics and the time evolution of the fluorescence emission after a pulsed excitation can be described by a monoexponential decay function according to: :F(t) = A e^ with A = the initial fluorescence intensity after the excitation and \tau = the decay constant (fluorescence lifetime). In contrast, autofluorescent background consists of a multitude of individual emitters, which obey individual emission statistics. Accordingly, the time evolution samples a summation of numerous individual decay statistics and can be written as: :F(t) = \sum(A_1e^). The FIDSAM technique bases on a time correlated single photon counting (TCSPC) measurement and analyses the degree of deviation of a recorded fluorescence decay from a monoexponential behavior. This is achieved by fitting the recorded fluorescence intensity decay by a monoexponential decay function convoluted with the instrument response function. In a next step, the error value of the fitting procedure, \chi^2 , is extracted and its inverse value is multiplied with the original intensity value. This way, fluorescence signal, which originates from autofluorescence background and therefore exhibits increased error-values, is divided by a relatively large number, whereas fluorescence signal from target molecules exhibits small error-values around unity is divided by a small number and remains largely unaffected.


FIDSAM imaging

Typically, the FIDSAM technique is applied to a microscopy imaging. Whereas the measurement protocol equals fluorescence lifetime imaging microscopy (FLIM), the read-out parameter is different. In FLIM, the characteristic
decay constant A quantity is subject to exponential decay if it decreases at a rate proportional to its current value. Symbolically, this process can be expressed by the following differential equation, where is the quantity and ( lambda) is a positive rat ...
is derived, while FIDSAM analyses the shape of the fluorescence intensity decay via the error value. Due to this, FIDSAM is intrinsically not dependent on fluctuations of the fluorescence lifetime, which often occur due to the individual chemical surround of a chromophore, but on analysis of the isotropy of the contributing emitters in a given sample volume.


References

{{reflist Fluorescence techniques Microscopy