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).

Figure 1. In FCS, fluorescently labeled particles diffuse in and out of the confocal volume. The resultant signal fluctuations can be used to obtain quantitative measurements of physical parameters.
Figure 2. In FCCS, two differently labeled populations of fluorescent particles diffuse in and out of the confocal volume. The cross-correlation of the resultant signal fluctuations can be used to obtain quantitative measurements of co-localization and molecular interactions.

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

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

FCS Application Examples

  • RNA mobility in the cytoplasm
  • DNA mobility in the nucleus
  • Active transport versus hindered diffusion to detect binding and unbinding of proteins
  • Virus capsid disassembly, protein complex disassembly 
  • Lipid raft formation and interactions
  • Binding of small molecules to membranes (e.g. Ligand binding)
  • Binding equilibria for drugs and other low molecular weight ligands

FCCS Application Examples

  • Quantification of interactions: protein-protein, protein-ligand, lipid-protein, protein RNA, etc…
  • Kinetics of oligonucleotide cleavage by restriction endonucleases, e.g., to monitor apoptosis
  • Evaluation of liposome coating DNA (lipoplex) for gene therapy
  • Viral assembly (e.g., capsid and receptor) or disassembly

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!

FCS and FCCS are available for LSM 980 and LSM 880 microscopes. For more information on how you can add FCS/FCCS to your LSM please contact your local representative or email us at info .microscopy @zeiss .com
 

Interested in learning more about how researchers are using FCS and FCCS?

Dr. Finn Cilius Nielsen with his lab members Àngels Mateu-Regué (left) and Lelde Kalnina (center).

Click the button below to see how Dr. Finn Cilius Nielsen and his team at the Center for Genomic Medicine, Denmark, are using FCS and FCCS to probe the molecules that control protein levels in cells.

Dr. Chris Richards, director of the Light Microscopy facility and associate professor of chemistry, University of Kentucky.

Click the button below to see how Dr. Chris Richards at the University of Kentucky is using in vivo multiphoton FCS to quantify cerebral blood flow with high spatiotemporal resolution.