Applications Leica TCS SMD FCS

FCCS facilitates the analysis of molecular binding.

Interactions are directly evident by the presence of a cross-correlation between two spectrally distinct channels. Furthermore, the ratio of free versus bound fraction in equilibrium reactions can be quantified. FCCS measurements are conducted with the FCS wizard.

Example
Caspase6, a downstream effector of the TNF-dependent apoptosis pathway, dimerizes upon activation (shown in the graph). A GPF-labeled construct and an mCherry-labeled construct were expressed in HeLa cells. 50% of the resulting double-labeled dimers (grey box) are detected in the cross-correlation signal. FCCS thus proves the active form of Caspase.

We thank Prof. Dr. Roland Eils and Dr. Joel Beaudouin, Bioquant, University of Heidelberg, Germany.

The SMD FCS wizard is a special part of the confocal software LAS AF which guides the user intuitively through the whole FCS experiment.

The combination of FCS and APD (Avalanche Photo Diode) imaging provides a fast and efficient way to map concentrations in a cell or another structure.

FCS measurements are point measurements. They deliver local information about single spots within the sample. To get concentration information over a hole image, FCS and APD imaging can be combined using the same detector. This enables scientist to calibrate the APD image intensity by the particle number obtained from the FCS measurement. In this way expression levels which are displayed as an image intensity can be quantified.

The acquisition of concentration mapping is well supported in the SMD FCS wizard.

Example: Concentration mapping of TiF1a-GFP in HeLa cells. The calibration of APD image was performed with FCS point measurement. ROIs for FCS measurements are shown as black crosses. The concentration was calculated from autocorrelation curves (red and blue graphs). A spatial map of intracellular concentrations is obtained. The correlation curves yield information about the particle motility at the measured points.

We thank Dr. Matthias Weiss, Dr. Jedrzej Szymanski, and Nina Malchus, DKFZ, Heidelberg, Gemany

The SMD FCS wizard is a special part of the confocal software LAS AF which guides the user intuitively through the whole FCS experiment.

The wizard supports

• the sequence of the image acquisition
• the optimization of the FCS measurement conditions
• the positioning of FCS measurement spots in 3 dimensions (xyz or xzy)
• the performance of the FCS measurement

Thereby, hardware settings for imaging and FCS are independent from each other. This comprehensive software protocol allows the user to control all aspects of the experiment.

Special features

• Nested FCS time series at multiple FCS measurement spots allow setting up complex experiments
• For the first time, highly precise positioning on membranes and vesicles is possible: FCS measurement points can be defined in xyz image stacks
• During a running FCS time series, control images can be acquired automatically, Any change of the specimen during an FCS time series becomes visible
• Permanent data quality evaluation is possible due to continuous display of online correlation curve, count rate and counts per molecule.
• The FCS wizard can be used for other point measurements as well: FCCS, FLCS, FLCCS or gated FCS
• APD imaging is supported

Leica TCS SMD FCS Single Molecule Detection System

The Leica TCS SMD FCS  is a dedicated system for reliable acquisition and analysis of FCS  and FCCS data. Both methods focus on quantitative analysis of transport and binding processes, in vitro and in vivo. The SMD FCS wizard optimizes FCS and FCCS measurement conditions and allows the setup of a complex FCS and FCCS time series at multiple measurement positions.

Typical FCS and FCCS applications

a) in solution (biochemical assay)

• Nucleic acid hybridization assays
• Protein and peptide interactions with liposomes
• Protein-protein and ligand-receptor interactions
• Characterization of photo-physical properties of fluorophores and autofluorescent proteins.

b) in live cells or vesicles

• Ligand-membrane receptor and protein-DNA interactions
• Concentration mapping
• Oligomerization: protein and lipid clustering
• Transport processes and signal transduction
• Lipid phase transitions
• Diffusion and binding of synthetic markers, nucleic acids, and proteins in different cell compartments