MICROSCOPY AND IMAGING FACILITY
LIGHT AND FLUORESCENT MICROSCOPY
Microscopes have been a primary scientific tool since the first curious investigator noticed the magnifying properties of water or glass and put those properties to use in answering scientific questions. Microscopy has played a major role in the study of cells. This direct visual examination has revealed much about the morphology of cells and tissues. In recent years, developments in microscope optics, dyes, staining protocols, and preparation techniques have helped reveal even more about the structure and function of cells and tissues.
The optical system of a conventional light microscope can be separated into three parts. These are a condenser lens, an objective lens, and oculars. Light from a tungsten filament bulb illumination source is directed through a condenser lens which focuses it onto the specimen. The light passes through the specimen and into an objective lens. It is this lens that resolves the fine detail present within the specimen. It projects the magnified image to a fixed position behind the lens where it is further enlarged by eyepieces, called oculars. Light emerging from the oculars is focused to a point where the eye or camera can see the magnified image.
When excited by light radiation of varying wavelengths, some substances will emit a light of a longer wavelength. This phenomenon is called fluorescence. These substances, called fluorochromes, can be attached to proteins such as antibodies which can then be used to detect specific structures or molecules within cells and tissues by light microscopy.
In fluorescent light microscopy, the normal tungsten filament light source of the microscope is replaced by a high pressure mercury vapor or xenon bulb which emits a very intense beam. This high energy light is passed through optical excitation filters which allow only a specific wavelength range of light to reach the specimen. This light excites the fluorochromes in the tissue on the microscope stage which respond by emitting a higher wavelength light energy. The emitted light is then passed through an optical barrier filter which only allows passage of a wavelength range of light specific for the fluorochrome in use.
Leica DMR-HC Upright Research Microscope
A research quality microscope with brightfield, differential interference contrast (DIC), and epi-fluorescent capability for blue, green, and near ultraviolet excitation. Objectives from 1X through 100X are available. The microscope is an integral part of the image processing and analysis system. The microscope uses a QImaging Retiga 4000R camera with 2048x2048 pixel resolution.
Leica DM-IRB Inverted Research Microscope
A research quality inverted microscope with brightfield, differential interference contrast (DIC), phase contrast, and epi-fluorescent capability for blue, green, and near ultraviolet excitation. Objectives from 5X through 100X are available. The microscope is an integral part of the image processing and analysis system. The microscope uses a QImaging Retiga 4000R camera with 2048x2048 pixel resolution.
CONFOCAL LASER SCANNING MICROSCOPY
Confocal laser scanning microscopy was developed as an alternative to conventional fluorescent microscopy in order to obtain higher resolution images and produce three dimensional reconstructions of fluorescent specimens. In the typical confocal microscope, a scan generator is used to raster a highly focused excitation laser beam in the x-y axis which is focused by the objective lens onto a fluorescent specimen. The emitted fluorescent light is captured by the same objective and focused onto a photomultiplier detector through a dichroic mirror, or beam splitter. As the laser is scanned across the specimen, the presence and strength of the emitted fluorescent light signal is detected by the photomultiplier and converted into a digital signal which is translated into a pixel-based image displayed on a computer monitor. The popularity of confocal microscopy arises from its ability to produce blur-free images of thick fluorescent specimens at various depths. Confocal imaging rejects any out-of-focus information emitted from a specimen by the addition of a confocal aperture, or pinhole, in front of the photomultiplier so that only the region of the specimen that is in focus is imaged. This ability to reject out of focus light from above or below the focal plane enables the confocal microscope to perform depth discrimination, or optical tomography. The plane of focus (z-plane) can be controlled by a computer-operated stepper motor which moves the microscope stage and is capable of producing optical sections as thin as one tenth of a micron. Digital images from various depths in a specimen can be presented as a single optical section or, by the use of repeated scans with focal plane changes, as an extended focus view or processed as a 3-dimensional reconstruction image.
Zeiss 510 META Confocal Laser Scanning Microscope
The instrument is based on an Axiovert 200MOT inverted microscope. There are seven laser excitation lines to choose from from 4 lasers: 405nm from a UV diode laser, 25mW, 458, 477, 488, and 514nm from an Argon 30mW gas laser, 543nm from a Helium-Neon solid-state 1mW laser, and 633nm from a Helium-Neon solid-state 5mW laser.
There are 4 photomultiplier detectors: Two for normal confocal imaging, one for transmitted light imaging, and the META detector. The META detector provides for better multi-fluorescence imaging and separation of fluorochromes which could not be imaged together on other instruments. The META detector also allows better separation of background autofluorescence from fluorescent dye emission. The META detector has 32 channels of detection which allows for signal processing that can "fingerprint" any fluorochrome by means of a "Lambda Stack" (a spectral analysis of emission wavelength). This "Emission Fingerprinting" can then be used to separate dyes with overlapping fluorescent emission spectra that could not be separated by conventional confocal methods. This Emission Fingerprinting can even be applied to online imaging.
Objectives installed on this instrument: Plan-Neofluar 10x/0.30, Plan-Neofluar 25x/0.8 Imm Corr DIC, LD Lci Planapo 25x/0.8 Imm Corr (long working-distance, multi immersion), F-Fluar 40x/1.30 Oil C-Apochromat 40/1.2 W Corr, Plan-Apochromat 63x/1.40 Oil DIC, and Alpha-Plan-Apochromat 100x/1.46 Oil DIC. All objectives have differential interference contrast imaging capability.
The instrument is equipped with a motorized scanning stage with controller, two heated stage inserts with temperature control, and an objective heater for short-term live-cell imaging.
The software has been recently updated to the Zeiss ZEN 2008 Windows®-based software which is user-friendly and logical. In addition there are modules for Multitime and FRET for specialized applications.
Zeiss 700 Confocal Laser Scanning Microscope
The newest addition to the facility is a 2 channel confocal microscope specially designed for long-term live-cell imaging. It is based on an Axio Observer Z1 inverted microscope. There are four laser excitation lines from four solid-state lasers: 405nm, 488nm, 555nm, and 635nm.
Objectives installed on this instrument: Plan-Neofluar 2.5x/0.075, Plan-Neofluar 10x/0.30, LD Lci Planapo 25x/0.8 Imm Corr (long working-distance, multi immersion), C-Apochromat 40x/1.2 W (long working-distance, water immersion), and Alpha-Plan-Apochromat 100x/1.46 Oil DIC.
The instrument is equipped with a motorized scanning stage with controller. It also has the Zeiss Definite Focus controller for extremely precise focus control during long-term conditions where focus drift is a problem. A LSM 700 XL S1 incubation system enables precise temperature for the entire instrument and there is there is a heated stage insert for addtional temperature control and CO2 levels at the specimen stage environment.
The instrument uses the Zeiss ZEN 2009 Windows®-based software which is user-friendly and logical. In addition there is a Multitime module for specialized applications.
The roots of digital imaging can be traced way back to the 1960's during the time of NASA's lunar program. Likewise, the cold war influenced the development of digital imaging as high altitude mapping and surveillance became daily necessities for a cautious nation in dangerous times. The development of the modern computer has expanded the availability of this technology to anyone with a quality personal computer.
By its purest definition, digital image processing is the capture and modulation of images by digital means. Within the digital domain, a digital image is represented by discrete points (pixels) of defined brightness and spatial location. Manipulation of the brightness or spatial characteristics of these pixels by digital algorithms (digital filtering) modifies the image to suit the user. Image analysis is the process by which a digital image is probed numerically to produce non-pictorial results. Instead of modified visual images as in image processing, image analysis extracts numerical or graphical information contained within the digital image. Such information may be morphometric, such as size or shape, or the information may be densitometric, such as brightness or optical density.
Digital imaging has many uses for the investigator today. At the very least, macroscopic and microscopic images which are digitally obtained and stored can be printed, displayed, and transferred. Digital image processing allows for elucidation of specific parts of an image and image analysis provides quantitative data where it was not possible before. Image processing and analysis can be utilized on any image that can be visualized by a digital input device. Such devices include analog and digital cameras as well as scanners.
The image processing and analysis system is Macintosh-based. The software is Improvision’s server-monitored OpenLab which not only enables digital image capture from the microscopes in the facility, but also will accommodate users to work at their own computers throughout CMRC for off-line image processing and analysis. Digital input from the Leica microscopes is through QImaging four megapixel digital cameras. The cooled CCD camera has the capability to input color brightfield as well as fluorescent images into the computer. The software allows for digital imaging of microscopic images and quantitative analysis of those images for morphometrics and densitometry. The Openlab software network in CMRC is one of the largest of its kind in the United States. There are currently over 45 systems and off-line computers in the research facility that use Openlab.
The Digital Darkroom is a Macintosh-based system with the Adobe Creative Suite 2 (Photoshop, Illustrator, InDesign, ImageReady) and Microsoft Office Professional (Word, Excel, PowerPoint, Access) as the main software components. The system uses inputs from a Microtek ScanMaker 1000XL scanner and network connections throughout the medical center. Publication-quality photographs from digital files are produced on a Fuji Pictrography 4000-II printer.
DARKROOM The Microscopy and Imaging Facility maintains a complete "wet" darkroom in room C.263 containing a QPS x-ray developer for processing of films for biochemical and molecular analysis.