UCSD Cancer Center - Digital Imaging Services

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Microscopy - Services

DeltaVision Deconvolution Microscopy	Laser Scanning Confocal Microscopy
*Bioluminescence/Fluorescence in vivo* Imaging with Xenogen IVIS 200 system**	Image Analysis and Photo production & Interactions With the VisLab at SDSC
Fluorescence In Situ Hybridization and Chromosomal Painting	Cell Microinjection

DeltaVision Deconvolution Microscopy.

Golgi bodies in mammalian cells.

For this 3-D visualization, two different protein components of the Golgi bodies (green, red) along with the genetic material of the cells (DNA, blue) were imaged using deconvolution microscopy. Deconvolution microscopy was performed by Drs. Maria Aparecida Pinhal and Jeff Esko, Department of Cellular and Molecular Medicine at UCSD, and Brian Smith of the Shared Resource. 3-D volume rendering was performed by Dave Nadeau and Philip Fisher-Ogden at the VisLab of the San Diego Supercomputer Center using NPACI Scalable Visualization Tools.

The system captures digital images by optical sectioning at steps at ~ 0.1-.3 um through the sample, acquiring multi-probe images at each step. Equipped with a Mercury arc lamp, with our present filter sets it can image at four wavelengths, 380, 488, 568, and 647 nm. These wavelengths are useful for dyes such as DAPI (Hoechst), fluorescein (Alexa 488, GFP, YFP), rhodamine ( Texas red, PE, Alexa 555), and Cy-5 (Alexa 647). It is mounted on a Nikon inverted light microscope with infinity corrected lenses. lenses available include 10x, 20x, 40x (oil), 60x (oil) and 100x (oil.) Please consult the manufacturer's website for more info, at http://www.api.com/lifescience/DeltaVisionRT.html. Their website, if you follow the links, will guide you to numerous publications utilizing deconvolution microscopy and the technical specifications of the imaging system, including specs of the fluorescence filter sets the system has.

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."

Laser Scanning Confocal Microscopy

A Zeiss LSM510 confocal microscope (LSCM) is available in within the Cancer Center Shared Resource housed in CMM-East. One limitation to deconvolution microscopy is its use of wide-field illumination. Less intense, the light does less damage to fluorophores but cannot penetrate deeply into thick specimens, usually greater than 30 microns in thickness. Thus having access to a LSCM system provides complementary utility to investigators for imaging fluorescence-based imaging needs. The Zeiss LSM510 confocal microscope is set up on an Axioscope2 platform and is equipped with 4 lasers giving a total of 7 laser lines at 351nm, 364nm, 458nm, 488nm, 514nm, 543nm, and 633nm. The microscope has three confocal epi-illumination channels for simultaneous imaging of 3 fluorophores, and a transmitted light channel with individually adjustable pinholes for each epi-illumination channel. More than 3 fluorophores can also be imaged in the multi-track/sequential mode. These features make it suitable for a number of applications, including co-localization, fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET) and ratiometric measurements.

(See http://www.zeiss.de/C12567BE0045ACF1/Inhalt-Frame/C98CD5EF1EFAF4EEC1256AC5003148E9 for more information from Zeiss)

The IVIS 200 in vivo Bioluminescence/Fluorescence Imaging

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. An integrated fluorescence system (400–900 nm) allows easy switching between fluorescent and bioluminescent spectral imaging applications, while a 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 The instrument is equipped with 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 CCD, cryogenically cooled to –105° C (without liquid nitrogen), minimizes electronic background, and maximizes sensitivity.

(See http://www.xenogen.com/wt/page/ivis_200 for more information from Xenogen)

Image Analysis and Photo production & Expanded Interactions With the VisLab at SDSC.
Image analysis,3-Dimensional reconstructions, specialized quantitative computation, and time-lapse serial images are analyzed using a number of computational tools that are now available on the SGI and PC work-stations. The computers are networked to each other, to users within the Cancer Center, and to the San Diego Supercomputer Center (SDSC). With VisLab members Colin McGinn, Natalie Rubin, Cameron Chrisman, Philip Fisher-ogden, Dave Nadeau, Alex Decastro, Steve Cutchin and Mike Bailey, new utilities have been made available to us to facilitate data management and analysis. Examples are 3-D rendering software packages such as Mesh Viewer, and Volume Explorer, that support interactive viewing of multi-modal, multi-resolution volume data, and generation of volume rendered 3-D images from different perspectives .

Example 1 - Click on image to enlarge	Example 2 - Click on image to enlarge

EGFP Expression in Cardiac Cells The image shows EGFP fluorescence in neonatal rat cardiac myocytes following gene transduction. The raw microscope image is on the left and the resultant quantification of the fluorescence intensities by SoftWorx Data Inspector is shown on the right. The experiment was done by Drs. Mei Hua Gao and James Feramisco in conjunction with Dr. Kirk Hammond.	Sarcomeres in Cardiac Cells The image shows the fluorescent signal following immunostaining of alpha-actinin in adult rat cardiac myocytes. The image was captured by deconvolution microscopy using the linear response range of the camera and the intensities of the pixels were measured by the program SoftWorx. The experiment and imaging were done by Dr. Mei Hua Gao and Julie Sherman in conjunction with Dr. Kirk Hammond.
Other example images created with these tools can be found at http://vis.sdsc.edu/research/cancer.html.

Fluorescence In Situ Hybridization and Chromosomal Painting.
FISH and chromosomal painting are techniques important to cancer research. Working with Dr. Karen Arden in the Ludwig Institute for Cancer Research, we have been able to offer these imaging services. With the recent addition of an Applied Spectral Imaging system for enhanced chromosomal painting and analysis, we expect the use of this service to grow.

Cell Microinjection.
Cell microinjection allows us to introduce molecules into the cells. The microinjection service of DICM provides a unique, centralized facility for use by Center Members. The primary goal is to enable investigators to make use of a highly specialized technique that can complement traditional experimental approaches for the analysis of the function of gene products in living cells.

Training.
The Staff, including the Resource Leader, offer training and advice to all potential and current users. The training covers instrumentation, theory to provide the investigators with a better understanding of the possible uses of the Resource and, equally importantly, its limitations.