Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Hardware Aspects ZEISS
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2.2 Principle of Fluorescence Correlation Spectroscopy
Fluorescence Correlation Spectroscopy (FCS) analyzes the diffusion time of molecules and their
differences if they have bound together. This is done by fluctuation analysis of fluorescence-labeled
molecules within a well defined volume element. In most experiments, Brownian motion drives the
fluctuation. The volume element is the confocal volume defined by the excitation spot of a well focused
laser beam and the selected emission region defined by the properly aligned pinhole of the detection
optics (Fig. 2).
As such, the setup is the same as for a Laser-Scanning Microscope (LSM), but in the latter we are not
interested in the fluctuations but in the average intensity. As a matter of fact, what is the signal in FCS is
noise in the LSM. Since the fluctuations are more pronounced, if fewer molecules are in the volume, FCS
requires little molecule numbers (1-10). Whereas LSM is a scanning technique, FCS uses the beam is
parked in one spot.
The fluctuations are analyzed by treating the measured photon counts with mathematical methods called
correlation functions (Fig. 3). The amplitude of the function is inverse proportional to the molecule
number and the decay time gives the residence time of the molecule in the confocal volume and hence
its diffusion time. If the two interacting molecules are of different size, only the smaller one has to be
labeled using fluorescent dyes. This method is called auto-correlation. In this case the total auto-
correlation is the sum of the two different species. If the diffusion constants of both partners are similar,
they are both labeled with different dyes and cross-correlation is used. Often, photo physical processes
like triplet states impinge on the correlation function, but can be accommodated in the model. Then the
total correlation is the product of the single processes.
Fig. 3 shows a correlation curve for a two component translational diffusion with triplet. The two
components can be separated on behalf of their diffusion time (circles). Note that the contribution of the
two components adds up, whereas the contribution of the total diffusion process and the triplet multiply
to obtain the total correlation.
Because of the tiny size of the confocal volume and its nature, the measurement can be carried out, in
principle, in every area that is reachable by light and that is not smaller than an Escherichia coli bacterium
(approximately 0.2 fl). In particular, measurements can be done inside living cells or on cell membranes.
In order to be able to place the measurement volume at its proper place, it is advantageous to combine
FCS with powerful light microscopy, particularly a confocal LSM.
Fig. 3 Correlation functions