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Imaging cellular function using fluorescence microscopy

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Imaging cellular function using fluorescence microscopy. Dr. Paul Thomas Henry Wellcome Laboratory for Cell Imaging School of Biological Sciences. Henry Wellcome Laboratory for Cell Imaging. 4 widefield fluorescence microscopes: Invert with CO 2 incubator Invert with microinjector

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Imaging cellular function using fluorescence microscopy

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  1. Imaging cellular function using fluorescence microscopy Dr. Paul Thomas Henry Wellcome Laboratory for Cell Imaging School of Biological Sciences

  2. Henry Wellcome Laboratory for Cell Imaging • 4 widefield fluorescence microscopes: • Invert with CO2 incubator • Invert with microinjector • Upright with HiRes camera • Stereo with HiRes camera • 2 confocal microscopes: • Leica TCS SP2 UV • Zeiss LSM510 META • 1 multi-photon microscope: • LaVision BioTec TriMscope • with lifetime imaging detector • Analysis facilities: • Cell / organelle tracking • 3D reconstruction • Time series / 4D quantification • Image restoration / Deconvolution Tibialis anterior containing pericytes expressing YFP with muscle and collagen fibres imaged using Second Harmonic Generation

  3. Transmitted-light and Fluorescence imaging Eastern Sea Fisheries Illegally-scrubbed lobster pleopods Salmonella-infected epithelial cell Epicardial cells from an explant of neonatal heart Nitric oxide generation during bacterial killing in a macrophage

  4. Quantification – Intensity analysis and object identification

  5. Measurement of intracellular ion concentrations: Proton concentrations in endo/lysosomal compartments pH 3.5 pH 4.5 pH 5.5 pH 6.5

  6. Dynamic measurement of intracellular ion concentrations: Calcium concentrations during agonist stimulation Ratio F/F0

  7. Tracking objects: Autophagosomal movements during cell starvation

  8. Automated tracking: Cell migration during embryo development

  9. Recent addition: A multi-photon microscope with a mode-locked, Ti:Sapphire laser Advantage 1: Long wavelengths = Less scattering of light; therefore, deeper penetration of tissues Advantage 2: Short pulse width (~100 fs) = Increased statistical chance of two or more photons arriving in the same place at the same time - 2-3 long-wavelength photons = 1 short-wavelength photon Advantage 3: Excitation only at the focal point; i.e., no photodamage in out-of-focus regions Courtesy of Graham Ellis-Davies, Drexel University, Philadelphia

  10. Fluorescence Lifetime IMaging (FLIM) Typical fluorophore lifetimes ~2 ns FLIM provides a spatial map of cellular activity

  11. Services provided (charges subject to negotiation) 1. Self-run experiments on microsopes (training provided): Widefield, confocal and overnight timelapse experiments 2. Experiments run by laboratory personnel (no sample preparation, no analysis): All microscopes including multi-photon/FLIM 3. Experiments run by laboratory personnel, inc. sample preparation (no analysis): All microscopes including multi-photon/FLIM 4. All-inclusive service run by laboratory personnel: All microscopes including multi-photon/FLIM Contact: Dr. Paul Thomas, Director,The Henry Wellcome Laboratory for Cell Imaging,School of Biological Sciences,University of East Anglia,Norwich,NR4 7TJ.e-mail: p.thomas@uea.ac.ukTel: (01603) 592196Fax: (01603) 592250Imaging web-site: http://www.uea.ac.uk/bio/biobmi/

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