User manual LEICA TCS SP5 MP BROCHURE

[. . . ] Thanks to reduced absorption and scattering of the excitation light, two-photon and multiphoton confocal microscopes reach a penetration depth of about 400 µm. In the case of two-photon excitation, the dye is excited by the simultaneous absorption of two photons. Due to the non-linearity nature of two-photon absorption, the excitation is limited to the focal volume and the photobleaching outside the focal plane is reduced. Only inside the confocal volume the photon density is sufficiently high to allow two photon absorption by the fluorophore. In 2-photon excitation fluorescence emission occurs only on the focal plane. 1-photon excitation 2-photon excitation Multiphoton excitation performance improves with pulsed laser excitation in the NIR spectra. [. . . ] Middle right: SHG-signal in mouse heart muscle Bottom left: dorsal musculature of zebrafish 4 FLIM­FCS­FCCS With FLIM (Fluorescence Lifetime Imaging Microscopy) a variety of local parameters within cells or other structures can be measured, such as ion concentrations, molecule interaction, FRET and membrane potential. The researcher thereby acquires information about processes occurring on a molecular scale. Time-correlated single photon counting technology guarantees perfect exploitation of photons. The Spectral FLIM detectors which are located within the SP5 spectral scanner allow even to combine spectral and lifetime information adding a new dimension to your image data. Advantages of MP excitation are the deep tissue penetration, and less photobleaching outside the focus. Tuneable excitation wavelength allows the usage of a wide range of fluorochromes and fluorescent proteins FCS (Fluorescence Correlation Spectroscopy) is a method to measure concentration and diffusion rates quantitatively down to the single molecule level. The data can be used to analyze molecule interactions and transport processes within living cells as well as in vitro. This allows evaluating the dynamics of molecular systems and cellular structures. FCCS (Fluorescence Cross-Correlation Spectroscopy) is mainly used to measure molecular interactions between molecules of arbitrary size which are labelled with spectrally distinct fluorescent labels. In this context, MP excitation is of special interest because it often allows exciting both labels at one wavelength. This guarantees the same excitation volumes for both interaction partners leading to a better cross-correlation signal. 0. 9 0. 8 0. 7 0. 6 0. 5 0. 4 0. 3 0. 2 0. 1 0 10-7 10-6 10-5 10-4 10-3 lag time [s] 10-2 10-1 10 0 FCS: Autocorrelation from living cells expressing an EGFP fusion of a nuclear protein. The measurement lasted 50 sec and was acquired in photon mode with a time resolution of 1 MHz. FLIM: Zebrafish FLIM image (left) and corresponding intensity image (right) with nuclei, muscle tissue and yolk. The histogram contains the color legend for lifetimes. 5 Flexibility Dedicated objectives and microscope stands Due to the different applications in confocal microscopy, different motorized stands have been developed: the upright microscope DM6000, the inverted microscope DMI6000 for living cell experiments and the new fixed stage DM6000 CFS for electrophysiology. A full range of objectives HCX PL APO with outstanding performance has been developed (quality: class CS). All relevant parameters have been optimized for high resolution, large image field and excellent color correction in the wide spectral range from UV to IR (U-V-I). The new Leica HCX PL APO L 20 x 1. 0 water immersion objective has been specifically developed for the new DM6000 CFS microscope. This objective fits perfectly for electrophysiological studies and developmental biology. It offers high resolution, a large field of view and a high transmission in both visible and infrared regimes. To achieve a 1. 0 NA with a 20x objective means that large specimen can be imaged as a whole while still preserving details at confocal resolution. Leica objective HCX APO L 20x1. 0 Transmission HCX APO L 20x/1. 00 W 100 90 80 70 60 50 40 30 20 10 0 300 400 500 600 700 Wavelength 800 900 100 1100 External detectors NDD on DM6000 CFS microscope 6 External/non-descanned detection For detection, the internal spectral detectors in the scan head can be used. But given the intrinsic confocality of the method, excitation is limited to the focal plane. [. . . ] The resonant scanner of the Leica TCS SP5 works at 16000 Hz frequency in bidirectional mode. The system acquires 25 images per second with 512 x 512 pixels and at higher speed up to 250 images with 512 x 16 pixels. Dynamic processes with high time resolution can be imaged and measured, e. g. Ca2+waves. Tandem Scanner: By means of a motorized and computer controlled high precision device, a conventional and a resonant galvanotmetric driven scan mirror are exchanged into the proper position for scanning, while the scan electronics is switched simultaneously 7 Leica TCS SP5 MP Features Detection System · Specific stands · Specific objectives · 20 x water immersion objective · High efficiency photon collection · Combination of RLD and TLD · Up to 4 NDDs simultaneously Scanning System, Laser Ports · True single point confocal scanner · Up to 8k x 8k per image · Up to 250 images/sec · UV, Vis and IR in one system · IR specific port · EOM attenuation Drosophila larvae Order no. : English 1593102114 · LEICA and the Leica Logo are registered trademarks of Leica IR GmbH. FLIM ­ FCS ­ FCCS · APD (avalanche photo diode) for maximum sensitivity (quantum efficiency up to 80 %) for low light imaging and FCS · Dual channel FCS and FCCS · Both, spectral and external FLIM Advantages of Leica TCS SP5 MP · Deep penetration · Lowest sample damage · Real time imaging · High resolution mapping · Ready for integrated analytics Courtesy of Dr. [. . . ]