User manual LEICA TCS STED CW BROCHURE

[. . . ] Leica TCS STED CW The Fast Track to Superresolution · Freedom to choose ­ popular fluorescent dyes and proteins · Observe what´s inside ­ with confocal superresolution · Nanoscale imaging ­ devoid of mathematical artifacts · Upgrading, to STED ­ quick and affordable · Watch live cell dynamics ­ at the nanoscale! 2 Superresolution light microscopy is revolutionizing life science research with increasing speed. The charm of direct visual results from an intact specimen on the nanometer scale attracts scientists from all fields of biomedical research. In its early days, superresolution was only for biophysicists and optical specialists. Nowadays, it has become an indispensable method in many life science research institutes working with light microscopes. [. . . ] Nowadays, the use of these proteins as endogenous, highly specific markers has become a standard tool in light microscopy. The expression of a fusion protein allows the selective labeling of distinct structures without the need to permeabilize the cell and to incubate it in a dye containing solution. This reduces the effort of fluorescence labeling and offers the most direct way for live cell imaging. By offering excitation lines such as 488 nm and 514 nm in combination with the depletion line at 592 nm, proteins such as eYFP and Citrin can be imaged with the Leica TCS STED CW. This enables researchers to record in order to follow structural changes on the nanoscale ­ live!Standard fluorophores ­ for fixed and living cell investigations All researchers are attracted by the potential of superresolution microscopy. Still, they want to rely on their well proven standard procedures and labeling strategies. 8 Living Cell Imaging using Fluorescent Proteins Vesicle movement Confocal STED 3 m 3 m Confocal STED STED t = 0 sec 1 m t = 0 sec 1 m t = 18 sec STED STED STED t = 36 sec t = 54 sec t = 72 sec Time lapse experiment: movement of large dense core vesicles labeled with the fluorescent protein Venus inside of living PC12 cells. 9 Observe What's Inside ­ with Confocal Superresolution STED CW Features · Flexible STED-excitation: Ar Laser (488 & 514 nm) STED: Fiber laser 592 nm; intensity modulated by AOTF · XY-resolution (FWHM) < 80 nm (measured on Chromeo 488 nano-beads), depending on sample, embedding and staining · Integrated linear deconvolution · Z-resolution: confocal · Auto beam alignment of excitation and STED beam for long term stability · Vortex phase filter for maximum resolution · Available in combination with AOBS and dichroic systems · Simultaneous line sequential recording of STED and confocal possible · Recording speeds of > 20 frames per seconds with < 80 nm lateral resolution · Full range of SP5 features supported, exclusive 405/UV Life is three-dimensional ­ and many important events which scientists are interested in happen under the surface. Thus, there is no chance to get insights with imaging technologies which are limited to the area in direct contact with the coverslip. The superior optical sectioning of the true point scanning system TCS SP5 provides superresolution where you need it: deep in the sample. STED images of a 12 m thick drosophila larvae featuring a thick cuticula ­ no problem. This is the edge of confocal superresolution! Purely Optical The well-known saying "seeing is believing" expresses it best: The success story of light microscopy is directly connected to its direct delivery of information to the researcher about the investigated specimen. Breaking the diffraction resolution barrier by STED is the logical consequence of this very fact. STED is based on a well-thought-out interplay of fine optics and well understood photophysical processes of the fluorophore. And it is currently the method to achieve superresolution in a purely optical way. The Leica TCS STED CW microscope delivers superresolution pixel by pixel ­ independent of recording speed or the dye being used. Plenty of time can be saved since time-consuming data processing steps and complex algorithms are obsolete. STED microscopy requires one single frame to generate one superresolution image ­ in contrast to other localization or interference based concepts. This makes STED extremely robust against interframe-drifts and facilitates data handling due to their size. In addition, the experienced user can improve his data by applying image deconvolution on top of the STED recording. The processing steps are uncomplicated and the workflow is embedded into the Leica confocal software LAS AF. As in the TCS STED with pulsed laser beams, spatial overlay accuracy of excitation laser and STED "doughnut" remains crucial to get the best results. This is granted by the patented and software controlled alignment routine which adjusts the laser beams automatically, activated by a single mouse click and completed within a minute. [. . . ] The dynamic range of 6 orders of magnitude in combination with fast signal recordings make them the perfect choice to record detailed images ­ even in combination with the resonant scanner. AOBS Left: conventional beam splitting by dichroic mirrors requires many optical elements with fixed properties. Right: the AOBS® is electronically adaptable to all tasks. 16 Acousto-Optical Beam Splitter (AOBS) A critical element of incident light fluorescence microscopy is the beam splitter. Leica Microsystems has set the standard with the introduction of the AOBS. This optical device is a programmable deflection crystal, which very specifically directs narrow excitation lines onto the sample while passing the full emission onto the detection module. [. . . ]