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6 Operation Modes

tive brightness and the concentration ratio of the molecules to the measured PCH. The tech-

nique is also called ‘fluorescence intensity distribution analysis’, or FIDA. The theoretical

background is described in [9, 10, 15, 17, 21, 23, 24].

The PCH/FIDA technique can be extended for multi-dimensional histograms of the intensity

recorded by several detectors in different wavelength intervals or under different polarisation.

Multi-dimensional photon counting histograms have been shown to deliver a substantially

improved resolution of different fluorophores [16, 17]. With the large number of input chan-

nels available in the DPC-230 recording multi-dimensional PCHs is merely a matter of the

optical system.

Fluorescence Correlation

Fluorescence correlation spectroscopy (FCS) is based on exciting a small number of mole-

cules in a femtoliter volume and correlating the fluctuations of the fluorescence intensity. The

fluctuations are caused by diffusion, rotation, intersystem crossing, conformational changes,

or other random effects. The technique dates back to a work of Magde, Elson and Webb pub-

lished in 1972 [20]. Theory and applications of FCS are described in [8, 27, 28, 29, 30].

The (un-normalised) autocorrelation function G(

) of an analog signal I(t) and the cross-

correlation function, G

(

), of two signals I

(t) and I

(t) are

For photon counts, N, in consecutive, discrete time channels G(

) and G

(

) can be obtained

by calculating

∑

+⋅= )()()(

tNtNG

∑

+⋅= )()()(

2112

tNtNG

For a randomly fluctuating signal, I(t), the autocorrelation function G(

) assumes high values

only if the values if intensity values, I, at a given time, t, and at a later time, t+

are correlated.

Uncorrelated fluctuations of I cancel over the integration time interval. Similarly, G12(

) as-

sumes high values if the fluctuations in both signals, I

(t) and I

(t+

), correlate with each

other. The drop of G(

) and G

(

) over the shift time,

, shows how far the fluctuations are

correlated in time. The general behaviour of the auto- and cross-correlation functions is illus-

trated in Fig. 9.

I(t)

G( )

I1(t)

I2(t)

G12( )

Fig. 9: General behaviour of the autocorrelation function, G(

),of a signal I and the cross-correlation function,

(

), of the signals I

and I

1 2 ... 7 8 9 10 11 12 13 14 15 16 17 ... 69 70

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