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 Zeiss 510 META combines an expanded laser system with an advanced META detector 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 Zeiss META has 6 laser "lines" of excitation (405, 458, 488, 514, 543, and 633nm). The addition of the 405 laser allows for imaging DAPI and other blue-emission fluorochromes.
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. As an example, the 510 META can image GFP, CFP, and YFP at the same time with good imaging of all three dyes.
The Zeiss Windows-based software is comprehensive but is user-friendly and logical. Confocal imaging is expedited by the ability to save and reuse configurations in another session. There is an off-line workstation in the facility to analyze images.
The introduction of the electron microscope as a tool for investigators brought about a complete reappraisal of the microanatomy of biological tissues and cells. Because of the need to see things at higher magnification and greater detail, it became necessary to rethink conventional microscopy. Instead of using light to illuminate a specimen, which has a wavelength range of 365 - 800 nanometers and a resolution potential between 130 - 190 nanometers, an electron beam is used as the illumination source. An electron beam at a modest 50,000 volts has a wavelength of 0.0055 nanometers and a resolution potential of 0.27 nanometers. Current electron microscopes have the ability to resolve at least 0.14 nanometers point-to-point (as a point of reference, a human hair is approximately 20,000 nanometers in diameter). In order to focus a beam of electrons, it is necessary for electron microscopes to have a columnar tube at high vacuum and magnetic lenses to modulate the beam. In the early days of its application, the electron microscope became the preferred tool of anatomists and histologists and many previously unimaginable structures in cells were revealed. More recent developments in specimen preparation have come from biochemists and cell biologists who use the electron microscope to answer complex questions about cells and tissues. The two most common electron microscopes available are the transmission electron microscope and the scanning electron microscope.
The transmission electron microscope consists of an evacuated column with the source of illumination, usually a tungsten filament or lanthanum crystal, at the top. As the filament is electrically heated at high voltage, it emits negatively-charged electrons which are passed through a tiny aperture (pinhole). Electromagnets, placed at intervals down the column, focus the electrons, acting just as the glass lenses in a conventional or fluorescent microscope. The beam is transmitted through the specimen and focused onto a phosphorescent screen for viewing, exposing photographic film for conventional film recording, or digital camera for computer image capture. Transmission electron microscopes have the ability of magnifying a specimen over 1,000,000 times at extreme resolution.
JEOL 1200EX Transmission Electron Microscope - The instrument is capable of conventional transmission electron microscopy (TEM) as well as scanning-transmission electron microscopy (STEM). It is equipped with a side-entry motor-driven goniometer which allows for tilting of +/- 60 degrees and 360 degrees of rotation of the specimen. The range of magnification possible is 50 - 1,000,000X. The instrument is exceptionally good at producing quality low magnification photographs as well as high resolution, high magnification photographs. The instrument uses a twin diffusion pump configuration to obtain high vacuum and a tungsten filament for durability and reliability. The instrument has an accelerating voltage range of 40 - 120 kV. The microscope produces 3 1/4X4 inch negatives for photographic enlargement and is equipped with a Hamamatsu digital camera for digital image capture. Maximum resolution for conventional transmission electron microscopy is 4.5 angstroms point-to-point, for scanning-transmission mode is 15 angstroms, and in scanning mode is 30 angstroms.
DIGITAL IMAGE PROCESSING AND ANALYSIS
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 Improvisionís 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.
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.
The Microscopy and Imaging Facility contains all necessary support equipment for its instrumentation. This includes a Denton DV502 vacuum evaporator and two Sorvall MT2B ultramicrotomes.