Leica AF6000E – AF7000 Advanced Wideﬁeld Systems Living up to Life Live Cell Imaging Solutions That Grow with Research Needs ||| Flexibility and Modularity – Individually optimized system conﬁgurations – Available for upright, inverted and stereo microscopes – A range of application wizards, process and quantiﬁcation tools – A wide choice of hardware components ||| Upgradability – Systems that grow with changing research needs – From entry-level systems to high-end workstations for real-time studies – Convenient, on site upgrade process ||| Integration – Perfect harmony between hardware and software – Maximum system reliability – Easy user handling ||| One Platform – One software platform for all wideﬁeld research and confocal microscope systems – Ideal for imaging centers and multi-user facilities – Minimum training required 2 Biological and medical researchers are probing deeper into the secrets of life, and wideﬁeld ﬂuorescence microscopy is a key technology for imaging the living cell. The developmental and molecular processes of the cell can be visualized directly by wideﬁeld ﬂuorescence techniques to shed new light on the mysteries of life. Leica Microsystems’ new Advanced Wideﬁeld Systems provide the leading innovations to advance your own research quest and unlock the secrets of life. Users all over the world have beneﬁted from the speed, reliability, brilliant image quality and ease of use of the ﬂuorescence research systems from Leica Microsystems for many years. Together with leading scientists, Leica Microsystems has further developed its successful ﬂuorescence solutions into a modular Leica Advanced Wideﬁeld System line that can be customized to suit all live cell imaging requirements. The Leica Advanced Wideﬁeld System Series offers the right tool for your research; from routine imaging and documentation to live cell imaging workstations for complex real-time experiments. Regardless of the application, all Leica Advanced Wideﬁeld Systems utilize the same easy-to-use software platform, which is shared with the Leica Microsystems confocal systems. The Leica Advanced Wideﬁeld System Line consists of four modular product solutions, which can be customized for different levels of application. The modular design affords you the freedom to grow the system and to choose hardware and software components as needed. ❚ Leica AF6000 E – The entry-level software solution for high-quality ﬂuorescence imaging and documentation. Compatible with a wide range of manual and automated microscopes and cameras from Leica Microsystems. ❚ Leica AF6000 – The ﬂexible system for a variety of applications in ﬂuorescence microscopy and image analysis including time-lapse experiments, multi-positioning, z-stacking and deconvolution. ❚ Leica AF6500 – The high-speed ﬂuorescence system with all the functionality of the Leica AF6000 plus real-time controlled image acquisition for ultra fast 2D time-lapse experiments, ratio imaging, and triggering of peripheral hardware components. Dendritic cells infected with bacteria Green: GFP expressing enteropathogenic E. coli Red membrane staining: cy3 labeled anti cd44 antibody Courtesy of: Nadzeya Kramko, ZMBE, Infectiology Institute, University of Muenster and Ivan Liashkovich Institute for Physiology II, University of Muenster ❚ Leica AF7000 – The premium solution for highly demanding applications which offers full real-time control for fast 3D time-lapse experiments, TIRF, Fura2, FRET SE, deconvolution and peripheral triggering. EGF stimulated endocytosis Green: GFP tagged EGF-receptor Courtesy of: Vibor Laketa, Cell Biology and Biophysics Unit, EMBL Heidelberg 5-day-old transgenic zebraﬁsh larva Green: GFP – actin Courtesy of: Prof. Dr. Stephan C. F. Neuhauss, Institute for Brain Research, ETH Zurich 3 A System as Dynamic as Modern Research The new Leica Advanced Wideﬁeld Systems offer full ﬂexibility to adapt to your changing needs. No matter which modules and components you select to conﬁgure an individual system, you will beneﬁt from the utmost reliability and convenience. As many of the system components are designed and developed by Leica Microsystems, we offer unparalleled automation control due to complete integration of our hardware within the software platform. The basic Leica AF6000 E system can be upgraded with a software package to create a Leica AF6000 ﬂuorescence and imaging analysis system. Add a control unit to convert the system into a highend Leica AF7000 live cell workstation with full real-time control. “I need to perform high-resolution 3D-deconvolution microscopy in time while keeping the cells in optimal conditions. Leica Microsystems provides the right tool for my live cell research.” Due to the many different ﬂuorescence applications, all systems from the Leica AF6000 upward work exclusively with semi- or fully automated microscopes. Leica Microsystems technical experts perform the upgrade on site at your facility, which minimizes the inconvenience of system down-time. Dr. Eef Parthoens Department for Molecular Biomedical Research (DMBR), VIB - Ghent University, Ghent, Belgium >> 4 >>> Upgrade Options from Entry-Level to High-End Solutions Leica AF6000 E Leica AF6000 Leica AF6500 Leica AF7000 Main Characteristics Entry-level ﬂuorescence acquisition system, 2D time-lapse, multichannel overlay Fluorescence imaging and analysis system, high ﬂexibility (without real-time control) High-speed ﬂuorescence Dedicated live cell system, system, real-time control full real-time control of camera and one ﬁlter wheel Applications Fluorescence imaging and documentation General ﬂuorescence imaging, 3D time-lapse experiments, multi-positioning, deconvolution Functionality of AF6000, plus fast 2D time-lapse and fast ratio imaging, peripheral triggering TIRF, fast 3D time-lapse, Fura2, FRET, deconvolution, peripheral triggering Includes AF6000 E SW + PC AF6000 SW + PC AF6000 SW + PC + CTR6500 HS AF6000 SW + PC + CTR7000 HS Leica DM1000 Ergonomic system microscope Leica DM2000 Advanced system microscope Leica DM2500 Advanced system microscope, 100 W Leica DM3000 Advanced system microscope, motorized Leica DM4000 B Automated upright microscope for biological laboratory and research applications Leica DM5000 B Automated upright microscope for demanding research applications Leica DM5500 B Automated upright microscope for high-end research applications with motorized focus Leica DM6000 B Fully automated upright microscope for cutting edge biomedical research Leica DMI3000 B Manual inverted research microscope Leica DMI4000 B Automated inverted research microscope Leica DMI6000 B Fully automated inverted research microscope Leica M205 FA High-end stereomicroscope for ﬂuorescence imaging • • • • • • • • • • • • • • • • • • • • • • • • • • • • 5 Leica AF6000 E – Flexibility Comes First ||| Entry-level image acquisition system ||| Document your research ||| Combine with a wide range of Leica cameras and microscopes ||| 2D time-lapse and multi-channel overlay The Leica AF6000 E is the ideal entry-level solution for wideﬁeld ﬂuorescence microscopy. It is particularly suited to general ﬂuorescence imaging applications such as biological and cytomorphological examinations and for professional documentation of research results. The system is truly ﬂexible, as it can be teamed with both manual and automated Leica Microsystems microscopes. In addition, the Leica AF6000 E can be conﬁgured with the Leica M205 FA, Leica Microsystems’ automated stereo ﬂuorescence microscope. The system is compatible with many cameras in the Leica Microsystems range, which beneﬁt from full integration with the software. As with all systems in the Leica Advanced Wideﬁeld System Line, the Leica AF6000 E features an intuitive user interface and intelligent workﬂow guidance. Mouse kidney section Glomerulus Blue: DNA, DAPI Green: Alexa 488 WGA Red: Actin, Alexa 568 Phalloidin Image by: Dr. M. Beynon, Leica Microsystems, Milton Keynes, UK Leica AF6000 E with DM2500 upright microscope and Leica EL6000 external light source 6 Leica AF6000 – Truly Flexible for Fluorescence The Leica AF6000 provides all the necessary facilities for demanding ﬂuorescence applications. This ﬂexible, fully integrated system can be equipped with a large number of application packages and software modules. The Leica AF6000 application spectrum ranges from general ﬂuorescence work via multi-channel imaging to time-lapse experiments, multi-positioning and z-stacking. The system incorporates a wealth of standard features for image documentation, processing, quantiﬁcation and analysis. ||| Multi-dimensional 3D imaging ||| Live cell experiments ||| A range of application packages and software modules for further analysis Optional modules for FRET, advanced time-lapse, channel unmixing, colocalization and various deconvolution techniques will extend the analysis capability. COS1 cell culture Red: Galectin3-YFP Green: Caveolin1-CFP Courtesy of: Prof. Dr. R. Jacob, University of Marburg, Department of Clinical Cytobiology and Cytopathology, Marburg, Germany Leica AF6000 with DM6000 B upright microscope and climate chamber 7 Leica AF6000 – for Upright, Inverted and Stereo Microscopes Flexibility for a wide range of applications The Leica AF6000 is compatible with automated upright and inverted microscopes from Leica Microsystems. In addition, the Leica M205 FA ﬂuorescence stereomicroscope with FusionOptics™ is a perfect partner for the Leica AF6000, with applications in timelapse and 3D image capture in the world of developmental biology. The use of a motorized stage also enables documentation of images on several selected areas of interest in the specimens. Vascular anatomy of a zebraﬁsh embryo as revealed by GFP expression driven by the Fli-1 promoter. The Leica AF6000 can be combined with monochrome and color cameras as well as EMCCD cameras. The camera of choice for high-resolution ﬂuorescence imaging is the Leica DFC360 FX. Courtesy of: Dr. Brant Weinstein, National Institutes of Health, Bethesda, MD Leica AF6000 with M205 FA stereomicroscope and Leica EL6000 external light source 8 Leica AF6500 – Ultrafast 2D Time-lapse The Leica AF6500 is a high-speed ﬂuorescence system that allows real-time control of a camera and a ﬁlter wheel as required for fast 2D time-lapse studies. Automated research microscopes, both upright and inverted, from Leica Microsystems are the ideal platforms for a Leica AF6500. Use the ratio imaging function to measure concentrations of second messenger substances such as Ca2+ at resolution over long periods of time. The system is also ideal for examinations of a wide variety of cellular processes such as exocytosis, transport processes within the cytoskeleton and membrane fusions. ||| Real-time 2D time-lapse experiments ||| Multi-dimensional 3D imaging ||| High-speed ratio imaging ||| Peripheral triggering C. elegans GFP expression Image by: Dr. Werner Wittke, Leica Microsystems, Wetzlar, Germany Leica AF6500 with DM6000 B upright microscope, EMCCD camera and Leica EL6000 external light source 9 Leica AF7000 – Real-time 3D Time-lapse Applications ||| Real-time 3D time-lapse experiments ||| High-speed FRET SE experiments ||| TIRF experiments The Leica AF7000 leaves nothing to be desired in wideﬁeld ﬂuorescence imaging. This system combines the ultimate in speed, reliability, scope for experiment design, analysis facilities and operational convenience. Achieve excellent results – fast! This extremely versatile and fully integrated system allows realtime control of all necessary hardware components for complex live cell studies, such as the camera, ﬁne focus devices and up to four external ﬁlter wheels. In addition, it offers the possibility to observe live cells using TIRF. With the Leica AF7000 you are expertly equipped for the research tasks of today and tomorrow. The Leica AF7000 effortlessly performs fast 3D time-lapse experiments. It is also fully capable of performing FRET SE, TIRF, as well as fast Fura2 measurements. Trigger signals can be transmitted to peripheral components via four channels. Vice versa, trigger signals can be received in up to four channels from external components and used to start experiments. Co-migration of the zebraﬁsh mechanosensory lateral line placode (green) and innervating axons (red) at 36 hours post fertilization. Live embryo is a double transgenic carrying neuronal-speciﬁc and placode-speciﬁc constructs (NBT:dsRedExpress and claudinB:mGFP, respectively). z-stack 200 slices/80 μm. Processing steps: dye separation and deconvolution. Courtesy of: Dr. Darren Gilmour, EMBL, Heidelberg, Germany Leica AF7000 with DMI6000 B inverted microscope, climate chamber, CO2 controller and SuperZ Galvo focus 10 TIRF (Total Internal Reﬂection Fluorescence) is the ideal technique for the study of cellular membranes and their environments. With automatic laser adjustment to maintain constant TIRF penetration depth, the Leica AM TIRF MC offers maximum ﬂexibility for TIRF applications with multiple ﬂuorophores at wavelengths of 405 nm, 488 nm, 561 nm and 635 nm for multi-color applications. The individual laser lines are coupled and controlled quickly and precisely via AOTF (Acousto-Optical Tunable Filters), achieving a switching time of only 1 ms. The Leica AF7000 is the base workstation from which to upgrade to the Leica AM TIRF MC at a later date. COS1 cell culture, Wideﬁeld Red: Galectin3-YFP Green: Clathrin-CFP COS1 cell culture, TIRF Red: Galectin3-YFP Green: Clathrin-CFP Courtesy of: Prof. Dr. R. Jacob, University of Marburg, Department of Clinical Cytobiology and Cytopathology, Marburg, Germany “Cell Biology is a fascinating field of research with a wide range of applications. My equipment needs to grow with the increasing number of demanding applications – Microsystems just like the Leica Microsystem imaging solutions.” Prof. Dr. R. Jacob University of Marburg, Department of C Clinical Cytobiology and Cytopathology, Marburg, Germany Leica AM TIRF MC – visualize life‘s secrets with true multi-color TIRF 11 Design Your Experiment – with Intelligent Modules Mitosis observed DIC H2A-YFP No mitosis observed DIC H2A-YFP t1 Common to all Leica Advanced Wideﬁeld Systems, Leica Microsystems has developed an easy-to-use, intuitive software interface that guides you through every step of the work process. Incorporating the latest ﬁndings in ergonomic design and efﬁciency research, the software enables you to deﬁne experiments and analysis swiftly and effortlessly. t2 t3 t4 t5 t6 From the simple control of the microscope functions to the highly complex confocal systems, all the ﬂuorescence research systems are built on the same software platform. This saves time when you ﬁrst start to work, getting your results fast. All settings are stored so experiments can easily be repeated. Log ﬁles and image parameters give detailed information on all actions performed during an experiment. A context-sensitive help function is available whenever needed. Support for complex analysis procedures comes in the form of speciﬁc wizards. For example, FRET SE offers application-oriented help and explanations. ||| Mark t7 NIH 3T3 mouse ﬁbroblast cells expressing H2A-YFP. Two positions of a Mark and Find experiment. Courtesy of: Dr. Constantin Kappel, DKFZ, Heidelberg, Germany and Find – Activation of the live image during the experiment – Change the cell positions while the experiment is in progress ||| Live Data Mode – Change the course of an experiment “on the ﬂy” – Online generation and graphical display of measurements – Flexible deﬁnition and combination of various experimental routines – Peripheral in/out triggering of experiments; control of manipulators or peristaltic pumps ||| 3D Visualization – 3D rendering and dynamic user interaction of volume – Compile multi-channel 3D movies Colocalization module: 2D-cytoﬂuorogram 12 ||| Colocalization – Visualization and quantiﬁcation of colocalized ﬂuorochromes – Statistic determination of the degree of colocalization ||| Dye Finder – Elimination of cross-talk between ﬂuorochromes with a proprietary unmixing algorithm – Differentiation between dyes with overlapping spectra ||| Deconvolution – Removal of haze and blur by reassigning scattered light – Fully integrated deconvolution algorithm using adaptive, calculated or supplied PSF Wideﬁeld image stack Deconvolved image stack Cerebellum (nervous system), ﬁxed sample, 40 μm thickness Courtesy of: Dr. Fulvio Florenzano, S. Lucia Foundation, Rome ||| FRET – – – – SE Application module for the acquisition and analysis of FRET specimens Correction of cross-talk Online display of FRET image and FRET graph FRET Sensitized Emission and FRET ratio ||| Fura2 – Determination of Ca2+ concentrations for intracellular signal processing and transport processes – Ca2+ quantiﬁcation according to Grynkiewicz formula – Online generation of measurement curves and cleaning of background ﬂuorescence ||| Well Plate Acquisition – Predeﬁned patterns for 6, 12, 24 and 96 microplates – User-deﬁnable patterns – Combination with time-lapse, z-stacking and tile scan Fura2 software module: increasing Ca++ concentration in the cell by adding Bradykinin Ratio intensity chart of Fura2 time-lapse experiment 13 Optimum Conditions for Live Cells Live cell experiments need optimized hardware components. Key requirements include fast or real-time image recording, temperature stability, minimum exposure of specimens to light and highly sensitive recording techniques for weak ﬂuorescence. For successful studies and high-quality results, all parameters have to be optimized for the specimens being analyzed and the experimental conditions. ||| Wide choice of cameras from Leica Microsystems, supplemented Leica Microsystems offers a wide choice of hardware components and accessories for individual conﬁguration of your research system. The modules can be combined in many different ways. The full integration of all automated functions with the software ensures perfect interaction of components in everyday use. ||| Wide choice of microscope stages ranging from manual through by highly sensitive EMCCD cameras. The Leica DFC360 FX is the premier high-resolution, high-performance ﬂuorescence camera recommended for the majority of Leica AF7000 applications. The Leica DFC360 FX offers overlapping mode for fast real-time studies, excellent signal-to-noise ratio, 40 MHz readout rate for real-time exposures and a highly sensitive CCD. motorized 3-plate stages to scanning stages. Extensive range of accessories for live cell examinations including climate chamber, cell chamber, various manipulators, CO2 controller, incubators. ||| ||| Fast external ﬁlter wheels and ﬁlter sets from Leica Microsystems with extremely short switching times. ||| Leica EL6000 external light source with high-speed shutter for minimum light exposure. ||| Leica TIRF objective for high spatial and temporal resolution in studies of dynamic processes inside or close to the cell membrane. ||| SuperZ focus for ultra-fast and highly precise z-stacking. High-Speed Components for the Leica Advanced Widefield System Line Time-lapse Multi-channel overlay Camera control Leica AF6000 E ● ● ● Z stack Motorized XY stage Deconvolution External ﬁlter wheels ● 1 FW* Leica AF6000 ● ● ● ● ● ● ● 1 FW* Peripheral triggering SuperZ Galvo ﬁne focus Pifoc ﬁne focus TIRF ●● available, high-speed ● available, non high-speed 14 not available Leica AF6500 ●● ●● ●● ● ● ● ●● 1 FW ●● 2 In, 1 Out Leica AF7000 ●● ●● ●● ●● ● ● ●● 4 FW ●● 4 In, 4 Out ●● ●● ●● *Leica CTR5500/6000/6500 required Leica External Fast Filter Wheel for high-speed excitation, attenuation and emission control. Leica STP6000 Smart Touch Panel for complete and convenient control of the microscope. Leica DFC360 FX high-resolution camera for fast ﬂuorescence imaging. Leica EL6000 external light source with high-speed shutter for ﬂuorescence excitation. Leica HCX Plan Apo objective with 1.46 numerical aperture for TIRF applications. SuperZ Galvo focus for ultra-fast and highly precise z-stacking. 15 Leica Microsystems – the brand for outstanding products • Life Science Research Division Leica Microsystems’ Life Science Research Division supports the imaging needs of the scientific community with advanced innovation and technical expertise for the visualization, measurement and analysis of microstructures. Our strong focus on understanding scientific applications puts Leica Microsystems’ customers at the leading edge of science. • Industry Division The Leica Microsystems Industry Division’s focus is to support customers’ pursuit of the highest quality end result by providing the best and most innovative imaging systems for their needs to see, measure and analyze the microstructures in routine and research industrial applications, in materials science and quality control, in forensic science investigations, and educational applications. • Biosystems Division The Biosystems Division of Leica Microsystems brings histopathology labs and researchers the highest-quality, most comprehensive product range. From patient to pathologist, the range includes the ideal product for each histology step and high-productivity workflow solutions for the entire lab. 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