Fluorescence Correlation and Cross-Correlation Spectroscopy (FCS)
Two systems are available at the Keck Center for FCS measurements - Becker & Hickl FLIM system coupled to the Zeiss 780 with two-photon illumination; ISS Alba system with one-photon illumination.
FCS is such a sensitive analytical tool because it observes a small number of molecules (nanomolar to picomolar concentrations) in a small volume (~1μm3). Florescence correlation spectroscopy (FCS) is an experimental technique that that measures fluctuations in fluorescence intensity caused by the Brownian motion of particles or molecules. Brownian motion is the random motion of particles/molecules suspended in a fluid that results from collisions with other molecules in the fluid. The initial experimental data is presented as intensity over time but statistical analysis of fluctuations makes it possible to determine various physical and photo-physical properties of molecules and systems. When combined with analysis models, FCS can be used for quantitative measurements of diffusion coefficients, hydrodynamic radii, average concentrations, kinetic chemical reaction rates, and single-triplet state dynamics.
The system configuration for FCS:
Confocal imaging system: ISS Alba V 2 channels
- Two SPCM-AQR-15 APDs
- Multi laser lines dichroics for FCCS or dual-color FCS
- Individual pinholes control for dual-color FCS optimization
- Nikon TiE and 60X / 1.2 NA water objective
Laser sources: 1p
- ISS diode 375-nm laser
- Fianium super-continuum laser lines at 448, 488, 514, 561, 635 nm
Data acquisition: ISS VistaVision software
- Count mode, time tagged mode and time resolved time tagged mode
- Time tagged mode allows resampling of the ACF calculation
- Time resolved time tagged mode takes both FLIM and FCS
- Support FCS acquisition at selected multi locations and in time series
Data analysis: ISS VistaVision software
- Online calculation of the ACF curve by the multi tau method
- Provide many built-in fitting routines for both 1p and 2p PSFs in 2D or 3D
- Allow users edit fitting equations to customize or define own models
- Process as many as ACF curves at the same time and allow global fittings through linking the same parameters in different ACF curves
- Provide utilities to easily export data and results
Lifu Wang, David Brautigan, Microbiology