See beyond Structure with Fluorescence Correlation Spectroscopy
More and more often, your research needs require looking beyond the structure of fixed samples to examining live samples in as close to physiological conditions as possible. Laser scanning microscopes have evolved to provide the fast and gentle imaging conditions that are required for these measurements and are capable of capturing high resolution images of fast-moving living samples. While there is a wealth of information that can be gained from these structural datasets, did you know that laser scanning microscopes are also capable of looking beyond structure to the molecular and mechanistic details of the sample? This can be easily accomplished with ZEISS laser scanning microscopes through the spectroscopic techniques of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS).
Advantages of FCS
FCS allows for the analysis of concentrations and mobility of fluorescently labeled “particles” such as DNA, proteins, and ribosomes. Compared to other techniques that are commonly used for measuring these parameters, such as fluorescence recovery after photobleaching (FRAP), FCS requires lower laser power, so is less damaging to the sample, and importantly provides access to information about sub-populations, rather than an ensemble average over multiple populations or compartments.
- Access to data that is otherwise not obtainable
- Data on molecular level
- Identification of specific sub-populations of one labeled particle
FCS Application Examples
Advantages of FCCS
FCCS allows for the measurement of dynamic co-localization, molecular interactions, and enzymatic reactions. Compared to common methods for assessing co-localization, such as fluorescence resonance energy transfer (FRET), interactions that occur at greater distances than are required for FRET (> 10 nm) can be measured, and there is greater freedom in fluorophore selection. Additionally, FCCS can easily be used to examine interactions between fast moving particles, which can be challenging with FRET as it is better suited for slower moving or static structures.
- Can be used for vesicle transport, assembly, and disassembly
- Large molecules and complexes: interaction even at > 10 nm distances
- FSCS: spectral cross-correlation spectroscopy with 32 channel GaAsP can provide additional information
FCCS Application Examples
How to use FCS and FCCS in your research
With the FCS and FCCS acquisition modes for ZEISS ZEN software, you can easily record and analyze FCS and FCCS data through a fully integrated, user friendly platform you are already familiar with from your confocal imaging.
- Switch quickly between confocal imaging and FCS acquisition.
- Easily image with multiple positions, and use a variety of sample carriers.
- Use built in curve fitting during the experiment or easily reanalyze the data after the experiment.
Use your existing detectors to measure:
- Mechanisms of transport
- Molecule mobility
- Molecule density (concentration)
- Molecule size
- Reaction rate
- Kinetics and more!