Microscopy and Imaging Shared Resource

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Equipment

Multiple fluorescence, confocal and automated microscopes allow high resolution microscopy required for visualizing molecules within intracellular compartments and tissues. They also enable digital documentation of histochemical, immunofluorescence, and in-situ hybridization experiments. Live automated imaging of cells or tissues is possible on confocal and widefield microscopes equipped with 37°C/CO2 chambers.

A microinjection system on an inverted microscope allows direct cellular injections of peptides, proteins, and plasmids. The behavior of microinjected cells can then be recorded using brightfield video microscopy.

The MISR supports image acquisition and analysis with Metamorph Image Analysis software and stand alone workstations as well as acquisition systems. Writable DVD/CD-ROM and external hard drives are available for image storage and transfer. Automated collection of digital images such as image montaging are possible with a motorized stage. Image analysis macros facilitate automated or semi-automated quantification of cell or tissue features.

Details of services and available equipment is listed below. A price list is available in Research Building room W407 or by request.

Conventional Light Microscopy

• Nikon E600 Fluorescence Digital Microscope System
  For routine viewing or multispectral imaging in bright field or fluorescence. Phase contrast, and DAPI, FITC, Texas Red, Cy5 multicolor imaging is available, for example.

• Olympus AH2 Vanox Digital Microscope System
  For routine viewing and imaging in bright field or fluorescence and equipped with a color CCD camera.

• Olympus IX71 Inverted Digital Microscope System
  For routine viewing and imaging in bright field or fluorescence and equipped with a color CCD camera.

• Nikon Eclipse TE-300 Inverted Timelapse Microscope System
  For live cell imaging of mutant GFP, YFP and RFP chimeras as well as fluorophore conjugated antibodies for recognizing extracellular epitopes. DIC optics and 3D reconstruction and deconvolution are available.

  Multi-well chambered, time lapse imaging using bright field or fluorescence microscopy is superb for following cell migration or cell division within a tissue culture environment.

• Zeiss IM-35 Inverted Microscope System with Eppendorf Microinjection System
  For routine phase, Hoffman, contrast and time lapse imaging.

• Nikon SMZ-1500 EPI-Fluorescence Digital Stereoscope System
  For stereoscopic visualization of tissues expressing GFP or RFP. Otimized for monitoring of invasion/metastasis of tumor cells in tissues. Motorized Z-stage acquisition allows for 3D image collection.

Total Internal Reflection Microscopy

• Olympus IX81 Total Internal Reflection Microscope
  For imaging plasma membrane in contact with the coverglass chamber slide.  Five laser line imaging capacity, with DualView for simultaneous imaging in two colors (GFP, RFP, CFP, YFP, far red).  TIRF and interference reflection microscopy (IRM) can be carried out sequentially. IRM permits visualization of cell contacts in living cells.
  
  The TIRF microscope (TIRFM) utilizes an optical phenomenon called Total Internal Reflection (TIR), which occurs when light travels from a higher to a lower refractive index medium at an angle greater than the critical angle.  Beyond this angle of total reflection, the electromagnetic field of the incoming/ reflected light extends into the Z direction and is termed the evanescent field.  One property of an evanescent field is that the magnetic strength of the field decreases exponentionally with distance, and so its effects extend only a few hundred nanometers into the second medium.  The portion of the specimen within the evanescent field, usually the cell membrane, can be excited to emit fluorescence which consequently can be seen or recorded.  TIRFM allows enhanced imaging of cell-surface proteins by substantially reducing background fluorescence from inside the cell.

Confocal Microscopy

• Olympus Fluoview-FV300 Laser Scanning Confocal System
  Routine fixed imaging and live time-lapse microscopy of samples using an Olympus IX-70 inverted microscope is available. Live cell microscopy of cells expressing GFP, CFP, YFP or RFP can be accomplished with Biopteks chambers (closed or open). Three dimensional imaging over time is possible for simultaneous detection of two fluorophores and bright field differential interference contrast (DIC or Nomarski) or internal reflection microscopy (IRM) to detect cell structure or cell adhesion formation. Four color experiments are also possible by sequentially capturing and then merging DAPI, FITC, Texas Red and CY5 images of fixed samples.

Multiphoton Micrscopy

• Zeiss 510LSM/ META/ NLO live imaging, multiphoton microscope
  With configuration #23 / META NLO + VIS, Argon, Dual HeNe, META and 2 PMTs, stage top environmental chamber for CO2 and O2 control, TiSapphire laser and FLIM hardware, this microscope is capable of FRET (sensitized emission and fluorescence lifetime emission (FLIM)), multispectral imaging, FRAP, time lapse, and 3D imaging for studies of subcellular localization and dynamic protein interactions.

Video Microscopy with Microinjection System

• Zeiss IM-35 Inverted Microscope System with Eppendorf Microinjection System
  For routine phase contrast, the Eppendorf microinjection system allows direct cellular injections of peptides, proteins, and plasmids. The system also offers routine time-lapse capabilities.
  

Electron Microscopy

Limited technical assistance or training (by appointment, pj42@georgetown.edu) includes using the TEM microscope as well as sample preparation for conventional negatively stained specimens, thin-sectioned embedded samples and immuno-EM.
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Image Analysis & Deconvolution

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Other Equipment and Technical Support

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