User manual LEICA TCS SP5 MP BROCHURE

[. . . ] · Uncaging, photoactivation or photobleaching in a diffractionlimited volume The Leica TCS SP5 MP covers a wide range of imaging applications (multiphoton and one photon) by combining two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. fluorescence emission excitation fluorescence emission Energy diagram of fluorescence with 1-photon and 2-photon excitations 2 Applications The invention of multiphoton microscopy in the 1990's raised a tremendous interest and has become a widespread imaging method in the biological sciences since then. Meanwhile there is plethora of applications and publications involving multiphoton microscopy. It is now established as the method of choice for non-invasive deep-penetration fluorescence microscopy of thick highly scattering samples and has been used for a diversity of specimen, e. g. lymphatic organs, kidney, heart, skin and brain (slices as well as intact organs). [. . . ] 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. FLIM measurements can be performed with pulsed visible or MP lasers. 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. These stands are fully integrated in the LAS AF software. 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. [. . . ] Photomanipulation experiments (uncaging, photoactivation or photobleaching) will be possible at this focal plane by using the EOM. Tandem scanner All multiphoton applications, morphological and high-speed dynamic studies, are covered by the Leica the Leica Tandem Scanner TCS SP5: Two scanners ­ one conventional and one resonant ­ are combined in the system and enables to switch between both scanners ­ fully motorized and computer controlled. The conventional scanner is optimized for morphological studies ­ brain, tissues, and cytoskeleton ­ allowing high spatial resolution. 8196 x 8196 pixel images can be obtained in combination with a large field of view (23 mm ­ intermediate image plane). The resonant scanner of the Leica TCS SP5 works at 16000 Hz frequency in bidirectional mode. [. . . ]