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LSM 510 in Live Cell Imaging Welcome LSM 510 in Live Cell Imaging Welcome Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Content The Subject of Interest Scanning Strategies for fast Image Acquisition Multicolor Labeling Emission and Excitation Crosstalk Spectral dimension - Emission Fingerprinting - Excitation Fingerprinting - Online Fingerprinting Applications - Quantitative FRET Imaging - FRAP and FLIP - Photoactivation & Photoconversion Zeiss features for live cell imaging Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging The Subject of Interest In fact Cells are highly dynamic Structures. The decision of a cell to proliferate or to underwent cellular suicide depends on dynamic analyses of a complex information network. Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Starting with the details – fundamental Questions • Which Ligand binds to the receptor? • Is molecule A changing the conformation of the complex of the molecules B and C? • How fast can a membrane associated protein reach its target structure in the nucleus? • Is there a binding secuence for that protein at DNA? What happens after binding? • Which nerve cell is responsible to react after a visual stimulus? • Which cells of an embryo are the progenitors liver cells? • How can the malaria germ enter a blood cell? Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging What are the specimens? Specimen Required equipment Cell culture Cell culture lab, cell incubation equipment at the microscope Tissue culture, tissue co cultures or Tissue culture lab, advanced incubation combinations of cell and tissue cultures equipment at the microscope (O2 control), with anorganic materials micro manipulation Living animals Animal housing, surgery equipment, special holding frames, vital function monitoring, micro manipulation Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging What are the dyes? Staining Principle Stained Components Direct or indirect Antibody Labeling Proteins, glycoproteins and polysaccarid structures at the cell surface Active uptake of fluorescent particles by Endosomes, lysosomes, phagosomes cellular transport mechanisms Dyes selective for the physical and Cellular membrane (DiI), DNA, endoplasmic chemical environment of a described reticulum, Golgi apparatus, mitochondria subcellular compartment Voltage dependent dyes Mitotracker red and green Ion sensitive dyes Ca++ imaging (Fura, Fluo4, Calcium Green) pH imaging (SNARF) Site specific expression of fluorescent Diverse number of proteins of interest proteins Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Scanning Strategies for fast Imaging Unidirectional Scan Fly Back Blanking, Zoom 1 Fly Back Blanking, Zoom 0.7 Fly Back Blanking, Zoom 1, Fly Back Blanking, Zoom 2, Rotation 45° Rotation 45°, X,Y Offset DSP controls Pixel Synchronized Ê AOTFs Ê Two Scanner Mirrors Ê Data Acquisition Bi-directional Scan Bi-directional Scan, Zoom 1 Bi-directional Scan, Zoom 1 Bi-directional Scan, Multitrack Rotation 45° Configuration Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging DSP : Scanmode Flexibility DSP controls Pixel Synchronized can • AOTFs S • Two Scanner Mirrors • Line Scan • Data Acquisition Spline With a motorized scanning stage n single XY frames can n patched together, for ROI Sca overview images of Tile Sca specimen with are exceeding Selective Excitation, a single field of Bleaching, Uncaging and view Data Acquisition from User defined ROIs Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Scan Mode: Online Crop Function • Use the Crop Function to adapt the scan field to the area of your interest • The scan field is free rotate able due to the two independent scanning mirrors • Any geometry from 1x4 to 2kx2k pixel is possible Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Scanning Strategies: Real Regions of Interest • Scanning real Regions of Interest (ROIs) is a feature of the LSM 510 requiring a fast laser switcher (AOTF, or AOM). • Up to 99 ROIs of regular or user defined shapes can be defined. • While scanning ROIs, the scan mirrors cover a field, neglecting all lines not including ROI pixel. A smart configuration of scan field rotation and ROI arrangement can save scan time. • Advantages: save memory, scan faster, prevent photo bleaching in neighbored cells, minimize photo damage. • Applications: Photo bleaching experiments (FRAP and FLIP) Uncaging (localized excitation, release of caged compounds). definition of ROIs full frame scan after for bleaching localized bleaching Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x512 Time resolution: 1s Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x100 Time resolution: 100 ms Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x32 Time resolution: 20ms Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Multifluorescence: The crosstalk problem FITC / Rhodamin double labeling: while simultaneous excitation and detection of both dyes a part of the FITC emission is detected in the Rhodamin detection channel (Emission Crosstalk). The problem can be solved by sequential excitation and emission detection. As a precondition the wavelength used for FITC excitation should not excite Rhodamin (no Excitation Crosstalk). Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Multifluorescence: Detection with Line and Frame wise Multi- Tracking The fastest switching between two tracks is available in with line wise switching at bi- directional scanning mode • The Configuration Control allows to define up to 4 different track for sequential dye excitation and emission detection. • The tracks can be switched line or frame wise. If line wise switching is active, no mechanical changes of optical elements between two tracks are allowed. Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging New: MultiChannel unmixing The solution: MultiChannel Unmixing Works on all LSM 510 and LSM 5 PASCAL with LSM Software Rel. 3.2 Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging New: MultiChannel unmixing The problem: excitation Crosstalk Crosstalk is the most often occurring problem in multifluorescence imaging. While emission crosstalk of dyes can easily be avoided by selective excitation with the Zeiss multitracking function, the use of dyes with overlapping excitation bands was hardly possible in non-META systems. Now the Zeiss ACE software is also available for the channel based LSM systems and solves the problem of excitation crosstalk in multi-labelling experiments. Crosstalk No crosstalk Æ Additional advantage: faster acquisition (single- instead of multitrack) Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Channel unmixing – Wide Field II FluoCells-Widefield raw data Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Channel unmixing – Wide Field II FluoCells-Widefield- unmixed Unmixed with MultiChannel ACE Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging The new way of detection in the scan head Based on the proven 510 concept META detector replaces one Single Detector PMT array with 32 elements Optical Grating for even dispersion Capture full emission spectra Fast electronic selection of PMT elements Truly confocal Adjustable pinhole (x,y, Ø) Field upgradeable 20.04.2004 -20- LSM 510 in Live Cell Imaging Emissipon Fingerprinting Lambda Stack Acquisition Separated Channel Image Linear Unmxing Extraction of Reference Spectra Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 5 Sytox Green and FITC in cultured fibroblasts Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 5 Sytox Green and FITC in cultured fibroblasts FITC Sytox Green overlay Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 6 CFP, CGFP, GFP and YFP Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting
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