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Microscopy and Image Analysis

Director:
Cristopher Bragg
bragg@helix.mgh.harvard.edu
Phone: 617-643-5754

Technician:
Tom Qin
tqin@partners.org
Phone: 617 - 726-0817

Pager: 617 - 339-6455
or online (http://ppd.partners.org, Pager ID: 22999)

 

The image analysis Core provides the NINDS research base with 1) hardware, 2) software, c) assistance and training services to acquire and analyze various types of images.

  • Hardware

For image acquisition, users currently have a choice between various instruments:

  • A Zeiss Pascal confocal microscope (room 6.025), based on a fully motorized Zeiss Axiovert 200, with an Argon (458/488/514 nm) and two Helium-Neon lasers (543 and 633 nm), fluorescence, bright-field, phase-contrast and Nomarski (DIC) optics, and the possibility to perform live cell imaging (temperature controller with custom chamber and heating stage).
  • A Nikon TE-2000 microscope (room 6.501A) with epifluorescence illumination and X/Y/Z Motorized stage
  • A Typhoon 9400 variable mode imager (room 6.501), for storage phosphor imaging, direct blue- (457, 488 nm), green- (532 nm) and red-excited fluorescence (633 nm) and chemiluminescence.
  • A Nikon Super Coolscan 9000 ED Scanner (room 6.501A), to scan stained histology specimens mounted onto microscope slides at up to 157 pixels/mm.
  • A Victor3 V plate reader (room 6.315), equipped with a 2-channel injector unit, for absorbance (UV/Vis), fluorescence, fluorescence polarization, time-resolved fluorescence, and chemiluminescence.
  • A Nikon TE2000-Perfect Focus System Microscope With Laser TIRF Digital Imaging (room 6.025) (the Olympus web site has an excellent introduction on what TIRF can achieve). This Nikon microscope platform has enabled us to seamlessly integrate a C1 confocal system, which includes manual switching hardware to allow directing of the LU-4 laser fixture light output to either the Ti-E inverted microscope TIRF attachment input or to the C1 confocal scan head. The Elements-C confocal software included in the TIRF system will allow image acquisition in TIRF, confocal, & widefield fluorescence imaging modes. Elements-C and the software modules we plan to purchase will control all motorized functions of the Ti-E microscope: z-focus, Perfect Focus System, motorized nosepiece, x-y stage, fluorescence filter turret, shutters for transmitted light & fluorescence, TIRF mirror angle, TIRF laser shutters, AOTF's & motorized transmitted light condenser. Additional Elements software modules will also, if needed, allow FRET/Ratio imaging and real time deconvolution.
  • Software

  • MCID Elite (room 6.501A): This system can perform general densitometry, analyze fluorescence (including dynamic fluorescence such as ratiometric and non-ratiometric ion imaging), gel/blot analysis, grain counting, 3D reconstruction, automated image stitching, tracer studies (such as local cerebral blood flow, glucose utilization), as well as limited stereology.
  • For more sophisticated stereological analysis, we purchased Stereo Investigator (Microbrightfield) (room 6.501A). This application is based on the unbiased stereology method and is currently considered as one of the 'gold standards' for obtaining unbiased counts from microscopic images. Stereo Investigator operates on our existing Nikon TE-2000 inverted microscope and is fully supported by both the Core personnel and MicroBrightfield (our service contract includes 'at-the-microscope' phone support as well as the ability for their support staff to remotely see the Core's monitor).
  • Investigation of structure often requires that optical or physical sections be made. Sectioning yield views of 2-D structure, but the third dimension is lost. MCID Elite contains extensive functions to measure the cross-sectional area, perimeter, and diameter of a target by counting the pixels within it (standard morphometry).  Common techniques for regaining knowledge of the third dimension are to perform standard morphometry on every section (exhaustive sampling), stereology and 3D reconstruction. In order to offer all these options to our user base, we acquired Improvision’s Volocity, which enables high-resolution volume rendering of 3D and 4D (i.e. 3D plus time) data sets, analyzes structure and function in 3D and 4D data, and identifies, measures and tracks objects in 2D, 3D and 4D.
  • The Elements-C confocal software included in the TIRF system allow image acquisition in TIRF, confocal, & widefield fluorescence imaging modes. Elements-C and its software modules control all motorized functions of the Ti-E microscope: z-focus, Perfect Focus System, motorized nosepiece, x-y stage, fluorescence filter turret, shutters for transmitted light & fluorescence, TIRF mirror angle, TIRF laser shutters, AOTF's & motorized transmitted light condenser. Additional Elements software modules will also, if enough users request them, allow FRET/Ratio imaging and real time deconvolution.
  • We recently pooled financial resources with the MGH Cardiovascular Research Center to introduce proteomics tools to the Neuroscience Center and purchase Phoretix 2D Evolution (Non Linear Dynamics), which provides various calibrations and functions to analyze 2D gels.

 

  • Services

In addition to providing the acquisition hardware and analysis software listed above, an essential function of the PI and Co-PI is to offer training, consultation and assistance during image acquisition and analysis. It is our experience that correct image analysis is often impossible because of poor study design and improper sample, section or image acquisition. Therefore, one of the missions of this Core is educational, in that image analysis should not be considered at the end of an experiment, but at the design stage. The technicians provide support with the practical use of the instruments, and are responsible for their maintenance.

 


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