In addition to the below Moores Cancer Center equipment, additional services are available at the
UC San Diego School of Medicine Microscopy Core, hosted by the Department of Neurosciences.
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Super Resolution Imaging Nikon A1R Storm Super-Resolution system allows users to obtain super-high resolution images below the normal diffraction limit for light microscopy. Super resolution is achieved by stochastic optical reconstruction from multiple frames obtained by rapid sequential excitation/deactivation of photo-switchable fluorescent probes. Only a small percentage of the fluorescent molecules are activated during any one cycle. Images are built from single fluorophore signals, in contrast to standard microscopy in which the image is the average of all fluorophore signals. This method produces images with resolution down to 20 nm in the x-y plane, and 50 nm in the z-axis – 10-fold greater resolution than standard microscopy.
Total Internal Reflection Fluorescence (TIRF) microscopy In TIRF microscopy an evanescent wave from light reflected at a critical angle selectively illuminates a field of view a few hundred nanometers at the cell-glass coverslip interface. This results in a high signal to noise ratio that is optimal for studying dynamic cell-surface events.
Laser Scanning Confocal Microscopy Optical sectioning is achieved by passing laser light through a pinhole that generates an image that is devoid of out of focus light typically found in wide field microscopy.
STORM (Scale bar is 1 microns)
STORM - higher resolution image. (Scale bar is 1 micron)
Mixed GFAP-vimentin intermediate filaments in U-87 MG Gliobastoma cells
U-87 MG Glioblastoma cells grown on coverslip bottom petridishes were fixed and stained with rabbit anti human GFAP, and mouse monoclonal anti vimentin primary antibodies, followed by Atto488 conjugated Goat anti rabbit, and Alexa 568 conjugated Goat anti mouse secondary antibodies. Comparison of confocal and STORM images of the same field are shown on top row above. Clear separation of GFAP and Vimentin staining is visible with STORM.
This YouTube video describes the principles of STORM:
The system captures digital images at Z steps of 0.1- 3 um through the sample. Iterative 3D deconvolution of the resultant wide field images and digital reconstruction results in greatly enhanced signal to noise that allows for unambiguous localization of gene products inside cells as well as in tissue specimens.
Image provided courtesy Muamera Mima Zulcic and Dr. Donald Durden.
Panel acquisition (7x 8) showing Retinal Neovascularization using anti-PECAM-1
Retina from a P7 neonate was isolated, flat-mounted, and labeled with anti-CD31 to analyze endothelial cell proliferation. CD31, also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), is a 140 kDa type I integral membrane glycoprotein that is expressed at high levels on early and mature endothelial cells, platelets, and most leukocyte subpopulation. PECAM-1 has various roles in vascular biology including angiogenesis, platelet function, and thrombosis.
For more information on deconvolution microscopy or laser scanning confocal microscopy, see the following articles accessible through PubMed:
The XENOGEN IVIS 200 Imaging System can be used to image both bioluminescence and fluorescence non-invasively in living animals, and to perform quantitative in vitro and in vivo assays using reporter cells tagged with a wide range of bioluminescent or fluorescent probes. The system uses a novel Xenogen technology in vivo biophotonic imaging to allow researchers to use real-time imaging to monitor and record cellular and genetic activity within a living organism.
Each mouse was transplanted intrafemorally to the right femur with 53,00 sorted cells isolated from fresh bone marrow biopsy of a multiple myeloma patient and transduced with GLF containing lentivirus. Bioluminescence signals were detected as early as 4 weeks post-transplantation.
Live IVIS image was taken 4 weeks after intrahepatical transplantation of H929 cells transduced with GLF lentivirus into neonates.
Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment in a Human Xenograft Mouse Model using IVIS200. Images provided courtesy of Christina C.N. Wu, and Dr. Dennis Carson
By passing laser light through a pinhole, out of focus light is removed resulting in optical sectioning of the specimen. Image is constructed by raster scanning of the specimen in XY plane from fluorescence picked up by the photomultipliers.
Fluorescence generated by excitation of fluorophores is captured by a CCD camera
Emission Spectra of recommended dyes and Bandpasses of the emission filters on the Nikon Upright microscope.
Transmitted light illumination of samples stained with chromogenic dyes
Users are strongly encouraged to discuss their plan of action with the microscopy staff before they start using the equipment. That way they are aware of which systems are best suited for their needs, as well as the proper protocol for sample preparation and staining.
The training covers instrumentation and theory to provide the investigators with a better understanding of the possible uses of the resource and, also its limitations. Once trained, users are granted permission to book and use the microscopes by themselves.
For infrequent microscope use, or for imaging material which requires a great deal of expertise, it may be more cost effective to let us assist you. Please contact us to schedule an assisted imaging session. On demand technical assistance with the microscopes is available during business hours should you need any more support.
Some techniques such as super resolution imaging may require special reagents and expertise. The microscopy core keeps some reagents on hand, or may be able to make some upon request. Please contact the microscopy staff to see what is available.
I addition to the above commercial software packages, the facility is greatly enhanced by collaborations with members of the San Diego Supercomputer Center (SDSC). These collaborations have truly changed the way we visualize and analyze 3-dimensional microscopy data sets. Using SDSC software and expertise, users of the Cancer Center's imaging facility have been afforded unique opportunities created by these remarkable groups of computer scientists.
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