Cell Imaging Protocols
The techniques and protocols that are regularly available to investigators are as follows:
Light Microscopy
Conventional tissue and cell evaluation are performed after fixation of tissue in either 10% neutral buffered formalin, paraformaldehyde, or 80% ethyl alcohol in glycerin/ acetic acid. Specimens are processed by dehydration, clearing, and embedment in paraffin wax. Tissue sections are cut at 4 mm and adhered to appropriately- prepared glass slides before application of hematoxylin and eosin or other histochemical stains. Cultured cells are examined by cytocentrifuge preparation or slide culture techniques with applied stains. Plastic embedded tissue is used for high resolution light microscopy via 1 mm sections stained with toluidine blue.
Immunofluorescence
Cells grown on glass cover slips are fixed for 15 minutes at room temperature with 4% paraformaldehyde in PBS. Tissues are fixed according to standard paraffin-embedding or cryostat-sectioning techniques. After rinses with PBS, the cells or tissues are incubated with a blocking solution consisting of 1% bovine serum albumin in PBS for three times five minutes, followed by incubation with a primary antibody for 30 minutes at room temperature. Immunoreactivity is detected with an affinity-purified secondary antibody conjugated to an appropriate fluorophore. The cells are rinsed, and coverslips are mounted on glass slides with Vectashield anti-fading solution (Vector Laboratories, Burlingame, CA). Tissue sections are rinsed and covered with a drop of Vectashield anti-fading solution, followed by a cover slip. Fluorescence signal will be observed on an Olympus BX50 light microscope equipped for phase contrast and epi-fluorescence, or with a Zeiss Axioscope
Images will be captured on 35 mm film with the PM-30 automatic photomicrographic system or captured digitally with an Optronics MagnaFire digital camera. For simultaneous localization of two antigens in the same cell, affinity-purified secondary antibodies conjugated to Alexa 488 and Alexa 568 will be used. Both fluorochromes will be visualized simultaneously with a multi-fluorescence cube (allowing visualization of up to three fluorochromes).
Confocal Scanning Laser Microscopy
Methods for cell or tissue fixation, processing, and immunostaining will be identical to those described for immunofluorescence. Immunostained preparations will be examined with a Bio-Rad MRC 1024ES confocal scanning laser microscope. Our system consists of a mixed gas krypton/argon laser with three excitation wavelengths of 488 nm, 568 nm, and 647 nm, allowing for the visualization of up to three different fluorochromes at the same time. This system provides for optimum image resolution and optical tissue sectioning for definitive antigen localization with superb spatial resolution. Software already implemented allows for histogram analysis of pixel intensity for fluorescence quantification, and the degree of co-localization for two fluorophores. "MicroVoxel", an interactive 3-D volume reconstruction software package for analysis and processing of confocal images is available on a dedicated workstation located in the same room. Direct color prints can be obtained from the confocal images from an Epson Stylus Color 850 printer attached to the system. The large image files typically generated by confocal scanning laser microscopy are stored on an attached 100 megabyte zip drive, followed by transfer to compact disks.
Laser Scanning Cytometry
Preparation of cells and tissues is similar as previously described for immunofluorescence. Cells on coverslips, or tissue sections mounted on glass slides are stained with fluorescent probes as desired. These include antibody staining, fluorescent dyes for DNA/RNA, and green fluorescent protein conjugates. The stained slides are placed on to the stage of the CompuCyte laser scanning cytometer (LSC), followed by automated stage scanning and excitation with an argon laser line (approximately 488 nm), or a helium/neon laser line (approximately 633 nm). The LSC is configured with multiple filter blocks and photomultiplier tubes to allow simultaneous assessment of three different fluorophores, or two fluorophores and forward scatter detection. An attached Donpisha true color ccd camera is used for image capture and documentation. The system software contains multiple analysis and processing capabilities. Acquired data and images are stored on 100 megabyte zip disks, followed by archival storage on compact disks. This system is ideal for investigations into cell cycle analysis, apoptosis and cell proliferation. The software is fully integrated to allow in depth data analysis.
Laser Capture Microdissection
An Arcturus PixCell II system is used for laser capture microdissection of cells. The system consists of an Olympus IX 50 research grade inverted light microscope with 10X and 20X objective lenses, and a solid-state laser diode (near-infrared, 812 nm). Tissue sections are cut from either paraffin-embedded or frozen tissues, and mounted on uncoated glass slides. The tissue fixation and processing protocol is essential to successful laser capture, and is established depending upon whether DNA, RNA, or protein is to be extracted. Investigators meet with Dr. Taatjes prior to tissue procurement to discuss tissue processing protocols. The slide containing the section is placed on to the microscope stage and held in place by a vacuum chuck. A "CapSure" cap containing a plastic transfer film is placed on top of the tissue section in the required area, and a laser pulse used to melt the transfer film into the section. Spot sizes ranging from approximately 6 mm to 35 mm can be used to isolate single cells, or entire regions. The cap containing the captured cells is then be transferred to an eppendorf tube containing the appropriate buffer. Analysis is then be performed using either gel mediated PCR or with the VCC TaqMan PCR device. The entire process is also recorded using an attached video camera and the PixCell II image archiving workstation. Each user has his or her own "login" password, and is able to set up capture sessions that are logically documented and archived. Images are stored on 100 megabyte zip disks, before archival onto compact disks.
Morphometry
Objective assessment of results of morphologic studies and data analysis for immunocytochemical studies are performed with Universl Imaging MetaMorph image processing and analysis software. This system consists of an Olympus BX50 light microscope (with phase contrast and fluorescence optics), and an Optronics MagnaFire digital camera. Images from sections or cells on glass slides can be captured by the camera mounted on the light microscope. Semiquantitative assessment of fluorescence or immunoperoxidase localization in cells and tissues are performed with this instrument's grey scale analysis mode or true color imaging mode with which reaction product can be thresholded and measured as a percentage of a standardized area or defined structure, for instance, an individual cell. Strict standardization of methods and uniformity of processing for experimental materials and controls is a prerequisite for this type of analysis to ensure accuracy of the numerical data. Alternatively, digitized images are analyzed with other image analysis programs, such as Photoshop (together with the ImageProcessing Toolkit plug-ins) and Image Tool. All of these programs have been successfully used for image analysis, and choice will be investigator initiated and dependent upon desired measurements. The Cell Imaging Facility Core now has a variety of morphometrically-oriented image analysis programs, allowing researchers to custom select their program of choice. Facility staff are available to consult with, and train users in the selected image analysis software program.
Image Processing and Analysis
A Dell dual 400 pentium processor workstation is available in the core for image processing and analysis. This system contains Adobe Photoshop (versions 4.0 and 5.5), The Image Processing Toolkit (Reindeer Games) as a Photoshop plug-in, Image Tool (from the University of Texas Health Science Center at San Antonio), Paint Shop Pro (Jasc), Adobe PageMaker, Confocal Assistant, and Microsoft PowerPoint. These software packages allow for diverse image processing and analysis requirements. Image montages are printed on an attached Fujix Pictrography 3000 video printer. This workstation also contains an AGFA StudioStar flat bed scanner for image or slide scanning. Although all of the imaging equipment in the core contains its own image processing and analysis software, the Dell workstation provides a common end point for image processing, freeing the imaging systems to be used for image capture, not analysis.
Electron Microscopic Methods
Tissue is cut into small pieces (approximately 1 mm3) and fixed by immersion in one-half strength Karnovsky's fixative (2.5% glutaraldehyde, 1% formaldehyde in 0.1 M Millonig's phosphate buffer, pH 7.2) for 45 min at 4°C. After 3 washes in Millonig's buffer (10 min each) the pieces are post-fixed in 1% osmium tetroxide in 0.15 M Millonig's buffer for 45 min at 4°C. The tissue pieces are dehydrated in graded ethanols and embedded in Spurr's resin. Ultrathin sections are cut with a diamond knife and picked up on copper mesh grids. After counterstaining with 2% ethanolic uranyl acetate and lead citrate the sections are examined in a JEOL 1210 electron microscope. For cultured cells, wash 2x2 min in serum-free media. Fix in 1-2% glutaraldehyde in Millonig's Buffer at 4°C for 1 hour, then post-fix in 1% OsO4 in Millonig's buffer at 4°C for 1 hour. Wash 3x15 min in Millonig's buffer and store at 4°C for up to a week. Dehydrate and embed in Spurr's resin.
