Applications Leica TCS SMD FLCS

The Leica TCS SMD FLIM is a dedicated system for FLIM measurements. These measurements reveal information about the direct neighborhood of molecules on the nanometer scale. The local conditions can determine folding states that influence the reactivity of molecules.

Furthermore, FLIM is used for binding studies (FLIM-FRET) and to identify measurement conditions and allows the setup of FLIM time series or FLIM stacks.

Special features

• Online display of Fast-FLIM-images, count rate and photon number allow to evaluate data quality during a running experiment
• A variety of pulsed lasers from UV via VIS to MP excitation can be selected according to the researchers needs
• Spectral FLIM detection combines lifetime and spectral information for the analysis of more complex fluorescence properties
• Leica TCS SMD FLIM systems equipped with PDM APDs from MPD are ready for an upgrade with FCS to an Leica TCS SMD FLCS system

Typical FLIM applications

Non-stained samples:

• Metabolic state characterization
• Cancer research: identification of modified cells
• Plant physiology: pathogen-host interaction
• Identification of chitin of lignin structures

Stained samples:

• Energy transfer (FLIM-FRET for molecule binding)
• Molecular sub-states (e.g. isomerization, protonation)
• Local concentration of ions and small ligands
• Environmental studies (viscosity, refractive index, pH)
• Dye separation
• Photophysical dye properties

Application Example (antennae of scale insect)
3D rendering of FLIM z-stack using autofluorescence. Color coded lifetime images were segmented according to chitin lifetime for isosurface rendering (green) and volume rendering of surrounding structures (red). Rendering was done using external software. Sample courtesy of Prof. Kees Jalink, NKI, Amsterdam, Netherlands

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