Microscopy Equipment and Services

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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Nikon A1R Confocal TIRF STORM Microscope (Moores Cancer Center Room 5365)

Principles of Operation

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.

Technical Information

  • Nikon Ti microscope with integrated autofocus (Perfect focus) that maintains focus when using glass slides, plastic culture dishes, or multiwell plates
  • Automated XY and Z stage (including a fast piezo Z)
  • Andor iXon+DU897, EMCCD (512x512 pixels with readout of 30 full frames per second) for low light applications such as TIRF, FRAP, and single molecule imaging applications such as STORM
  • Andor high speed sCMOS camera (Zyla 5.5 megapixel with readout of 100 full frames per second) for applications requiring a higher resolution and speed.
  • A1R hybrid confocal scanner incorporates two independent galvo scanning systems, a high-resolution (4096x4096) scanner, and a high speed resonant scanner (512x512) for rapid confocal scanning of live cells
  • LU4 four-laser AOTF unit with 405, 488, 561, and 647 lasers
  • Lumencore solid state light source for wide field illumination and fast switching
  • Plan Apo 10, 20 and 40X dry objectives, and 60 and 100X high NA (1.49NA) TIRF objectives
  • Provided with heating and carbon dioxide incubation for live cell work
  • The microscope is located in a BSL2 facility

Applications

  • Super resolution microscopy using stochastic optical reconstruction methods such as N-STORM, D-STORM, or PALM (Photo Activation Localization Microscopy)
  • Three-color D-STORM with conventional dyes Alexa488, Alexa568, and Alexa647
  • 3D super resolution imaging that allows accurate localization of sub-cellular components
  • TIRF microscopy of dynamic cell-surface events, such as endocytosis, exocytosis, focal adhesion dynamics, integrin and receptor mediated signaling,
  • Laser scanning confocal microscopy of fixed and live samples
  • Photokinetics, - simultaneous photo activation or photo bleaching (FRAP) and imaging
  • Forster Resonance Energy Transfer (FRET)
  • Generation of high quality 3D and multi color montages with the automated XYZ stage
  • High speed, or low light level imaging
  • Multi dimensional fast time-lapse imaging

Sample Images

Confocal

Confocal

storm 2 micron scale

STORM (Scale bar is 1 microns)

storm 1 micron scale

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.

Informational Videos about STORM and PALM

This YouTube video describes the principles of STORM:

These YouTube videos provide additional information:

DeltaVision Deconvolution Microscopy (CMME, Room 003A)

Principles of Operation

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.

Technical Information

  • Nikon TE200 inverted light microscope
  • Objectives with magnifications of 10x, 20x, 40x (oil), 60x (oil) and 100x (oil).
  • Automated XY and Z stage
  • The system is equipped with filters for imaging a wide range of fluorophores that include blue(DAPI), CFP, green (GFP, FITC or Alexa488), YFP, red (RFP, Rhodamine, PE, Texas red, Alexa555, Alexa568, Alexa594, etc), and two infra red channels, i.e., Cy5 (Alexa647), Cy7 (Alexa750)
  • High power (300W) Xenon lamp,
  • Sensitive and fast camera (Coolsnap HQ)
  • Sofworx 3D deconvolution and analysis software

Applications

  • Fluorescence imaging of fixed cells and thin tissue samples (less than 50 micron thickness)
  • Generation of multi color 3D montages following iterative deconvolution and stitching of multiple panels
  • The fact that it can acquire images of fluorophores with excitation and emission in the infra red region is an important feature when trying to correlate the observations made in live animals (using the IVIS whole animal imaging system) with events that are occurring at the tissue and cellular level. Whole animal imaging typically works best with fluorophores of long wavelengths because of their ability to penetrate deeper into tissue than those in the visible range, and are the probes of choice for in vivo imaging.

Sample Image 1

Confocal

Image provided courtesy Muamera Mima Zulcic and Dr. Donald Durden.

Sample Image 2

Image provided courtesy Muamera Mima Zulcic and 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.

Image provided courtesy Muamera Mima Zulcic and Dr. Donald Durden.

For more information on deconvolution microscopy or laser scanning confocal microscopy, see the following articles accessible through PubMed:

  1. "A three-dimensional structural dissection of Drosophila polytene chromosomes."
  2. "An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis."

IVIS 200 in vivo Bioluminescence / Fluorescence Imaging (Moores Cancer Center vivarium)

Principles of Operation

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.

Technical Information

  • Integrated fluorescence system (400–900 nm) allows easy switching between fluorescent and bioluminescent spectral imaging applications
  • Laser scanner provides 3D surface topography for single-view diffuse tomographic reconstructions of internal sources
  • Spectral imaging uses measurement data from a sequence of images filtered at different wavelengths, ranging from 560 nm to 660 nm, to determine the depth and location of a bioluminescent reporter
  • Excitation and emission filters for GFP, DsRed, Cy 5.5, and ICG in addition to a set of four background filters for subtraction of tissue autofluorescence
  • A 26 mm square back-thinned 16 bit 2048x2048 pixel CCD camera cryogenically cooled to –105° C minimizes electronic background, and maximizes sensitivity.

Applications

  • Non-invasive imaging of distribution of cells tagged with bioluminescent or fluorescent markers following injection in live animals over time
  • Localization of tumor or non-tumor cells in 3D in live animals, e.g. invasion and metastasis of tumor cells
  • Quantitative comparison of fluorescence or bioluminescence for tracking treatment efficacy over time

Sample Images

Confocal

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.

storm

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

(See more information from Xenogen)


Zeiss LSM510 Laser Scanning Confocal microscope (CMME Room 003C)

Principles of Operation

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.

Technical Information

  • The Zeiss LSM510 confocal microscope is set up on an upright Axioscope2 platform
  • Equipped with 4 lasers giving a total of 7 laser lines at 351nm, 364nm, 458nm, 488nm, 514nm, 543nm, and 633nm.
  • Three confocal epi-illumination channels for simultaneous imaging of 3 fluorophores with individually adjustable pinholes for each epi-illumination channel. It also has a transmitted light channel for DIC imaging. More than 3 fluorophores can also be imaged in the multi-track/sequential mode.
  • Multiple filters for each fluorescence channel allowing setup of new configurations for atypical fluorophores
  • Objectives include 10x water, 25x and 40x immersion objectives suitable for oil water or glycerol, 60x and 100x oil

Applications

  • Imaging of fixed cells and tissue specimens a few 100 microns in thickness
  • Tissue slices with high autofluorescence
  • co-localization
  • fluorescence recovery after photobleaching (FRAP)
  • fluorescence resonance energy transfer (FRET) and ratiometric measurements
  • Calcium imaging

Nikon Inverted Fluorescence Microscope (Moores Cancer Center Room 5317)

Principles of Operation

Fluorescence generated by excitation of fluorophores is captured by a CCD camera

Technical Information

  • Nikon TE300 inverted fluorescence microscope
  • 75W Xenon arc lamp
  • motorized Z stage for 3D image acquisition
  • Excitation and emission filters for DAPI, FITC, EGFP, Cy3, Texas Red, and Cy5.
  • Objective lenses include 4x, 10x, 20x, and 40X phase objectives in addition to 40X, 60X and 100x oil objectives Phase condenser lenses
  • 14-bit Q-imaging monochrome/color camera that can also be used to image cells stained with chromogenic dyes.

Applications

  • Examining expression of fluorescently tagged gene products such as GFP, RFP, mCherry, dsRed fusion proteins, in live cells, grown in tissue culture dishes using fluorescence and phase contrast microscopy
  • Fluorescently labeled fixed cells and tissue samples
  • 3D acquisitions as well as time lapse imaging of live cells
  • cells stained with chromogenic dyes in multiwell culture dishes.

Nikon Upright Fluorescence Microscope (Moores Cancer Center Room 2304)

Principles of Operation

Fluorescence generated by excitation of fluorophores is captured by a CCD camera

Technical Information

  • Nikon E600 upright fluorescence microscope
  • 150W Xenon arc lamp
  • narrow band-pass filters to image six fluorophores (DAPI, FITC, Cy3, Red, Cy5 and Cy7) in the same sample
  • Objective lenses include 4x, 10x, 20x, and 40X DIC objectives in addition to 60X and 100x oil objectives DIC optics
  • 14 bit SPOT Explorer mono/color dual camera that is more sensitive in the infrared region

Applications

  • Imaging both, fluorescence staining, as well as slides stained with chromogenic dyes
  • Imaging tissue samples stained with fluorophores with excitation and emission in the infra red region - an important feature when trying to correlate the observations made in live animals (using the IVIS whole animal imaging system) with events that are occurring at the tissue and cellular level. The existing filters are useful for imaging dyes such as Cy5, Cy5.5, Cy7, Alexa750

Spectra of Dyes and Filters

Emissions Spectra

Emission Spectra of recommended dyes and Bandpasses of the emission filters on the Nikon Upright microscope.


Nikon Teaching Microscope (Moores Cancer Center Room 2304)

Principles of Operation

Transmitted light illumination of samples stained with chromogenic dyes

Technical Information

  • Nikon E600 upright fluorescence microscope
  • Spot QE color camera
  • Multiple eyepiece ports for teaching or group
  • Objectives lenses include 2x, 4x, 10x, 20x and 40x dry, and 60x and 100x oil

Applications

  • Imaging Histology specimens stained with chromogenic dyes
  • Teaching and group observations

Services

Consultation

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.

Training

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.

Technical Support

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.

Nikon Elements software from Nikon

  • 2D and 3D STORM analysis module with automatic drift correction
  • Photokinetics and FRET modules
  • 3D rendering and measurements

Volocity 3D software from Perkin Elmer

  • Iterative (3D) deconvolution module
  • 3D rendering module
  • 3D registration and analysis module

Metamorph 2D

  • 2D measurements and analysis
  • 2D deconvolution

Softworx from Applied Precision

  • 3D deconvolution
  • 3D measurements and analysis

Image Pro Plus 3D analysis from Media Cybernetics

  • 3D analysis and measurements
  • 3D rendering

Custom Software Design

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.