Fluorescence intensity decay shape microscopy

Within the scientific study of a molecule's color, Fluorescence intensity decay shape microscopy (FIDSAM) is a fluorescence microscope technique, which utilizes the time evolution of fluorescence emission after a pulsed excitation to analyse the decay statistics of an excited chromophore. 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

−

t

/

τ

{\displaystyle F(t)=Ae^{-t/\tau }}

with

A

{\displaystyle A}

= the initial fluorescence intensity after the excitation and

τ

{\displaystyle \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

)

=

∑

(

A

1

e

−

t

/

τ

1

)

{\displaystyle F(t)=\sum (A_{1}e^{-t/\tau _{1}})}

.

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,

χ

2

{\displaystyle \chi ^{2}}

, is extracted and its inverse value is multiplied with the original intensity value.