Light Microscopy Core

About

For those requiring basic light microscopy the Core will help determine appropriate staining protocols and offer training and/or assistance in acquiring transmitted and fluorescent digital images with wide-field microscopes. Those requiring optical sectioning capability to better resolve their samples can receive training and assistance in the use of confocal and multi-photon microscopes. Whole slide scanning can be done in brightfield or fluorescence mode. Two Laser MicroDissection instruments are available for isolating small areas of tissue or even single cells. Total Interference Reflection Fluorescence (TIRF) microscopy is available for visualizing and quantifying cellular events localized at the basal plasma membrane. Live cell and tissue imaging can be done on both wide-field and confocal microscopes equipped with environmental chambers for heat/CO2 control.

In addition to the microscopy services, investigators also have access to Image Processing and Analysis workstations with a variety of advanced software for extracting quantitative data from their images. They can also generate images and movies for presentations, grants and publications.

The Core is home to 2 LI-COR Odyssey, laser-based scanning systems for gels, Westerns, in-cell Westerns, ELISA/FLISA, EMSA/Gel Shift, Protein Detection, etc. It makes use of the inherent sensitivity of the near-infrared spectrum, reduces autofluorescence, and enables direct detection (no film, darkroom, substrates).


Contacts

Judith A. Drazba, PhD

Judith A. Drazba, PhD
Staff Director, Imaging Core
Location:NB1-46
Phone:(216) 445-3760
drazbaj@ccf.org

Gauravi  Deshpande, PhD

Gauravi Deshpande, PhD
Imaging Specialist
Location:NB1-45
Phone:(216) 444-8045
deshpag@ccf.org

John W. Peterson, PhD

John W. Peterson, PhD
Project Staff
Location:NB1-40
Phone:(216) 444-8045
petersj@ccf.org

Ajay  Zalavadia, PhD

Ajay Zalavadia, PhD
Imaging Specialist
Location:NB1-45
Phone:(216) 444-8045
zalavaa@ccf.org

Services

The imaging specialists in the Core are dedicated to helping you obtain the best quality images that answer your scientific questions. We provide training on our microscopy instruments or we offer complete imaging services. We can assist with experimental design, image acquisition, troubleshooting, image analysis and the development of novel image analysis methods.

Widefield Microscopy
For imaging fixed samples, the Core provides microscopes equipped for transmitted light and fluorescence microscopy. Digital images are captured with high-resolution cameras. Large specimens can be imaged in their entirety with either transmitted or fluorescent light using an automated slide scanner or a stereomicroscope.

Live Imaging
Living specimens in culture (flasks, dishes or multi-well plates) can be imaged using transmitted light, phase contrast, DIC or fluorescence modes. Inverted microscopes are equipped with heat/CO2 incubators and motorized stages for capturing multiple time-lapse fields simultaneously. Widefield systems use high-resolution cameras in both transmitted light and fluorescence modes. Confocal/multiphoton systems use specialized photon detectors to capture high resolution fluorescence images.

Confocal Microscopy
"Optical sections" or "Z Stacks" of samples can be obtained on confocal microscopes. The stacks can be reconstructed with software that allows 3-D visualization and analysis. The microscopes are each equipped with multiple lasers for fluorescence excitation at a wide variety of wavelengths. This allows users to visualize multi-labeled components of a specimen simultaneously to compare the three-dimensional relationships between them. For example we can visualize fluorescently labeled proteins and compare the quantity and localization relative to other labeled cellular components.

Multiphoton Microscopy
This technology provides three-dimensional optical sectioning similar to that produced by a confocal, but because it uses longer wavelength light it is less damaging and can penetrate up to half a millimeter into tissue. It is ideal for imaging living specimens especially when deep imaging is required. It can be used for tissue sections employing simultaneous multiple IR wavelengths. With its combination conventional/resonant scanner the microscope can run in either slow scan mode for optimal spatial temporal resolution or fast mode (16,000 lines per second) for imaging rapid dynamic cell events.

Slide Scanning
Large specimens on slides that are labelled with either conventional histologic dyes or with fluorescent markers can be imaged in their entirety using high-resolution slide scanners.

Multispectral Slide Scanning
Whole slide multispectral imaging (up to 9 colors) for visualizing, analyzing, quantifying, and phenotyping cells in situ in Formalin Fixed Paraffin Embedded (FFPE) tissue sections and TMAs.

Observation of Cells under Shear Flow
Microfluidics for cell analysis under shear flow – mimicking physiological flow in human vasculature. Cells are first cultured in biochips. The chips (with the resulting cell monolayers) are mounted on the stage of a live-imaging microscope and attached to a pump that introduces the flow of solution containing cells. Interactions between the cells in the fluid and those immobilized in the chamber can be imaged and quantitated.

Laser Microdissection/Laser Capture
Precise excision and capture of cell groups, single cells or even parts of cells out of tissue sections or cultures. Such explants can be used for RNA, DNA or protein analysis.

Total Interference Reflection Microscopy (TIRF)
Fluorescently tagged molecules adjacent to a glass surface can we imaged with 70nm resolution. It is ideal for studying events in the plasma membrane of living cells with high axial resolution.

Stereomicroscope
Imaging of large unmounted samples with brightfield and/or fluorescence illumination.

Infrared Western Blot Scanner (Odyssey)
Scanning of protein gels, Western Blots and In-Cell Western Assays using infra-red illumination to increase sensitivity and reduce autofluorescence.

Image Analysis/Quantitation
Post-acquisition processing and image analysis using high-end workstations and powerful software packages. Services include:

  • Color segmentation of fluorescent and histochemical samples
  • Customized quantitation (area, diameter, integrated intensity, etc.)
  • Automated quantitation (batch processing) of large image stacks
  • Analysis of colocalization
  • Filtering and processing images for grants, papers, seminars, etc. See Equipment tab for software available
  • Training in the use of these software applications
  • Processing and/or analysis by Core staff on a case-by-case basis

Equipment












Grant Information

The Imaging Core provides a wide range of advanced imaging and scientific consulting resources in the areas of Light Microscopy, Electron Microscopy, Histology and Immunohistochemistry/FISH for investigators throughout Cleveland Clinic. The primary mission of the facility is to assist investigators in producing high-resolution images of cells and tissues using light and electron microscopy.

For those requiring basic wide-field light microscopy the Core will help determine appropriate staining protocols and offer training and/or assistance in acquiring transmitted and fluorescent digital images with wide-field microscopes. The Imaging Core has two Leica upright microscopes for capturing images from fixed specimens on slides in transmitted light (i.e. brightfield, phase contrast, differential interference contrast, darkfield) or fluorescence. They are equipped with high-resolution cameras for both black and white as well as color imaging. All microscopes are equipped with a full range of Chroma filters for imaging a broad range of fluorescent tags and proteins. They also are equipped with a complete range of objective lenses from 1.25X dry up to 100X water and oil immersion. Living specimens in flasks or culture dishes can be observed on inverted microscopes equipped with high-resolution cameras and motorized stages for capturing multiple time-lapse fields simultaneously. Images can be collected in both transmitted light and fluorescence modes. These microscopes can also be used for scanning multi-well plates in their entirety.

Those requiring optical sectioning capability to better resolve the fluorescence in their samples can receive training and assistance in the use confocal and multiphoton microscopes that allow for 3-D visualization and analysis. For confocal microscopy, the Imaging Core has three systems configured to accommodate both fixed and live cells/tissues. Each instrument is equipped with multiple detectors and lasers to allow for visualization of multi-labeled components of a specimen. Captured images reveal the three-dimensional relationships between them. For example, fluorescently labeled proteins can be imaged to compare the quantity and localization relative to other labeled cellular components. Since confocal microscopes are limited in their ability to view more than about 100 um into a specimen, the Core has a multiphoton system that is able to image 500um or more depending on the tissue. It can be used for tissue sections employing simultaneous multiple IR wavelengths. With its combination of conventional/resonant scanner it can run in either slow scan mode for optimal spatial temporal resolution or fast mode (16,000 lines per second) for imaging rapid dynamic cell events.

Total Internal Reflection Fluorescence microscopy is available to image fluorescently tagged molecules adjacent to a glass interface with 70nm resolution. It is ideal for studying events in the plasma membrane of living cells with high axial resolution.

The Core has two different laser microdissection systems equipped with integrated cutting lasers for precise excision and capture of cell groups, single cells or even parts of cells out of tissue sections or cultures without touching or contaminating them. Such excised samples can be used for RNA, DNA or protein analysis.

The Core has 2 LI-COR Odyssey, laser-based scanning systems for gels, in-cell Westerns, ELISA/FLISA, EMSA/Gel Shift, Protein Detection, etc. It makes use of the inherent sensitivity of the near-infrared spectrum, reduces autofluorescence and enables direct detection (no film, darkroom, substrates). The Core provides software so that users can open and analyze data on computers in their lab.

Large specimens (tissue sections) on slides can be imaged in their entirety with either transmitted or fluorescence light using a slide scanner or a microscope that produces tiled mosaic images. Dishes and organs can be imaged with the Leica MZ16 FA stereomicroscope. This instrument allows the sample to be seen in three dimensions and has a long working distance so that larger specimens can be arranged (dissected, if necessary) and imaged.

For cell analysis under shear flow – mimicking physiological flow in human vasculature – the Core has the microfluidics system of Cellix Ltd. Cells are first cultured in an incubator using biochips and flow chambers attached to a specially designed pump that provides the shear flow. The chips (with the resulting cell monolayers) are then attached to another pump that introduce flow into up to 8 separate tubes for up to 8 simultaneous assays. The chips are mounted on the stage of a live-imaging microscope for observation.

Image Processing and Analysis Workstations are available where clients can use leading image rendering and analysis software such as Image-Pro Plus (Media Cybernetics, Inc., Rockville, MD), Image-Pro 10 (Media Cybernetics, Inc., Rockville, MD), Volocity (3D Imaging Software) (PerkinElmer/Improvision, Inc., Waltham, MA), InForm Software (Akoya Bioscience,CA), HALO software (Indica labs, Albuquerque, NM), Photoshop (Adobe Systems, Inc., San Jose, CA), Huygens Software for Deconvolution (Scientific Volume Imaging b.v., Netherlands) to do:

  • Color segmentation of fluorescent and histochemical samples
  • Customized quantitation (area, diameter, integrated intensity, etc.)
  • Automated quantitation (batch processing) of large image stacks
  • Analysis for colocalization
  • Filtering and processing images for grants, papers, seminars, etc. See Equipment tab for software available.
  • Training in the use of these software applications
  • Processing and/or analysis by Core staff on a case-by-case basis
  • Highly multiplexed immunohistochemistry (IHC) methods (available in the Core) have significantly expanded the range of metrics collectable from tissue sections providing quantitative assessment of cellular phenotype and activity in a way that is similar to flow cytometry. More importantly, these new techniques provide tissue context and information about cell-to-cell interactions that is crucial for understanding intact tissue dynamics. The Vectra Polaris system provides whole slide multispectral imaging (up to 9 colors) for visualizing, analyzing, quantifying, and phenotyping cells in situ in Formalin Fixed Paraffin Embedded (FFPE) tissue sections and TMAs.

    Light Microscopy Core

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