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FLCS and gated FCS – The Method

FLCS measurements are performed with the Leica TCS SP8 SMD system.

Essentially, FCS can be performed with a continuous-wave laser. Using pulsed lasers allows even more sophisticated analysis possibilities, such as time-gated FCS or Fluorescence Lifetime FCS (FLCS). Both methods make use of the additional information obtained by the simultaneous measurement of the fluorescence lifetime.

A time-gate, for example, selects for analysis of only those photons that can be clearly attributed to the fluorescence decay. Suppression of scattered light (Rayleigh, Raman) from the detected signal allows an accurate measurement of concentration.

In FLCS each photon is statistically weighted as to how much it contributes to the FCS data analysis. It can be used to correct for detector artifacts or scattering contributions.

It even allows simultaneous monitoring of two dyes with completely overlapping emission spectra and with very similar diffusion coefficients by using only a single detector.

Sample: GalNacT2-GFP expressed in HeLa cells analyzed with FLCS. We thank Dr. Matthias Weiss, Dr. Jedrzej Szymanski and Nina Malchus, DKFZ, Heidelberg.

4. Comparison of FCS curve (no time filter) with FLCS curve (after time filtering)