Page "Fluorescence correlation spectroscopy" Paragraph 13

The typical FCS setup consists of a laser line ( wavelengths ranging typically from 405 – 633 nm ( cw ), and from 690 – 1100 nm ( pulsed )), which is reflected into a microscope objective by a dichroic mirror.

The laser beam is focused in the sample, which contains fluorescent particles ( molecules ) in such high dilution, that only a few are within the focal spot ( usually 1 – 100 molecules in one fL ).

When the particles cross the focal volume, they fluoresce.

This light is collected by the same objective and, because it is red-shifted with respect to the excitation light it passes the dichroic mirror reaching a detector, typically a photomultiplier tube or avalanche photodiode detector.

The resulting electronic signal can be stored either directly as an intensity versus time trace to be analyzed at a later point, or computed to generate the autocorrelation directly ( which requires special acquisition cards ).

The FCS curve by itself only represents a time-spectrum.

Conclusions on physical phenomena have to be extracted from there with appropriate models.

The parameters of interest are found after fitting the autocorrelation curve to modeled functional forms.