User manual LEICA TCS SMD DATASHEET

[. . . ] Leica TCS SMD Series Single Molecule Detection Platform for Obtaining Meaningful, Reliable Results: FCS, FCCS, FLIM, FRET, FLCS, Gated FCS Protein diffusion in polymer matrix 1, 0 normalized autocorrelation G() 0, 8 0, 6 Long polymers: Normal diffusion 0, 4 Short polymers: Sub-diffusion 0, 2 0, 0 1 2 0, 01 0, 1 1 10 100 Lag time [ms] 3 · All in one ­ a single platform to study the dynamics of life · Global control by full system integration · Quantification with maximum content · Dedicated application wizards for fast, reproducible work · Immediate results by intelligent hardware and software presets Leica Design by Christophe Apothéloz 2 4 Molecular interactions, such as protein complex formation, protein-DNA or ligand-receptor binding are extremely significant for modern biology (1, 2). The specific identification of interaction partners and quantification of binding parameters are crucial for the understanding the biological, chemical, and physical processes in live cells. This information is necessary for goal-oriented development of agents that influence biochemical reactions on a molecular level, which is essential in pharmaceutical drug design and biomedical research. Leica TCS SMD Series Quantify Life! Single Molecule Detection (SMD) and analysis is an elegant way to examine dynamics and interactions inside cellular systems. This includes the quantitative characterization of biochemical reaction kinetics and equilibrium on a molecular level. [. . . ] Instead, a distribution of times is observed, which can be described by an exponential decay function. The characteristic time constant of this decay, the fluorescence lifetime, is in the range of a few picoseconds (10-12 s) to several tens of nanoseconds (10-9 s). This lifetime is a characteristic parameter of each fluorescent dye that may change with its micro-surrounding or its conformational state. Lifetime information probes the molecular environment for its composition, such as ion concentration, pH, lipophilicity or the binding to other molecules. FLIM combines lifetime measurements with imaging: lifetimes obtained at the pixel-level are color-coded to produce images. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its nanoenvironment. This way an additional dimension of information is obtained. Laser pulses Principle of FLIM data acquisition and analysis: 1. Repeated measurement of the time between laser pulse and fluorescence photon at each pixel 2. Calculation of a histogram of photon counts over arrival time after the laser pulse 3. The amplitude reflects the total number of photons, the time constant is called the fluorescence lifetime 4. Display of lifetime image using a false-color look-up table Emitted photons 15 Spectral Freedom with SP FLIM PMTs The Spectral Detection Module 1 2 3 Prism Sliders Detector FLIM SP FLIM PMTs, which are integrated into the SP module of the scan head, ensure optimal adjustment to experimental conditions and removal of autofluorescence. Software-controlled mirror sliders in front of the detector select the wavelength range of interest. This gives the freedom and flexibility to choose the spectral detection range for FLIM. 3 1 2 SMD FLIM wizard: complex experiments easily run Using the SMD FLIM wizard the user defines all relevant parameters for FLIM acquisition, such as scan speed and format or acquisition time of single FLIM images. These settings are transferred to and automatically used by SymPhoTime Software. The SMD FLIM wizard offers a variety of FLIM scan modes to set up more complex sequences of FLIM data acquisition : FLIM volume stacks give information about the lifetime distribution in tissues or small organisms. With FLIM time series the researcher can follow dynamic changes of fluorescence lifetimes, especially in live cells or tissues. A new dimension of knowledge ­ FLIM lambda stacks: SP FLIM detectors are used for automated acquisition of FLIM lambda stacks, i. e. , FLIM image series at defined bands of the emission spectrum. Lifetime emission spectra are especially useful for characterization and identification of autofluorescence or new chromophores, for better separation of dyes with similar properties, and for identification of conformational states and aggregation of chromophores. Automated brightness control provides maximum data reliability: this unique feature generates images with pre-defined brightness. It ensures that the photon statistics of all recorded images are consistent. Automated brightness control can be used for volume and lambda stacks. Intrinsically it compensates for a fluorescence intensity decrease caused by photo-bleaching or light absorbance in deeper sample sections. FLIM 2 FLIM Advantages · Immune to effects such as: ­ Concentration fluctuations caused by diffusion, or photo-bleaching (without photo-conversion) ­ Shading in thick samples ­ Fluctuations of excitation intensity ­ Light source noise · Internally calibrated FRET · Unperturbed conditions in unstained samples 14 3D reconstruction of FLIM z-stack Complementary technologies available in LAS AF · FRET SE Wizard · FRET AB Wizard 16 Host ­ Pathogen Interaction ­ Identification of Invading Hyphae in Tomato Fruit Intensity Image [Cnts] 700 0 Average Lifetime [ns] 2, 97 0, 4 Chloroplasts Fungal hyphae Putative parenchyma 15 1. [. . . ] Intensity spectra (3) and lifetime spectra (4) show strongly overlapping, non-separable species. Spectral information with fluorescence lifetimes allows disentangling a complex mixture of (auto-)fluorescent species (i. e. Lambda series of three samples, a donor-only control, a co-transfection as negative control and a GFP-mCherry tandem as FRET sample (2). Detection window free of autofluorescence or acceptor fluorescence ranges from 485­545 nm (constant average lifetime) (3). [. . . ]