A spot of a minimum size in the specimen is illuminated and a n image is formed by scanning the specimen. The image of each spot is directed through a pinhole stop in an intermediate image plane. As a result, only light from the focal plane can reach the detector (a photomultiplier). All other (out-of-focus) planes are blocked out. This results in an "optical section". The images are then stored electronically and displayed on a monitor.
A series of optical sections can be recorded by moving along the z-axis each time an image has been recorded. Such a z-series permits the electronic reconstruction of the three-dimensional structure using suitable computer programs.
Conventional microscopes create images with a depth-of-field at high power of 2-3 micrometers. By contrast, confocal microscopes create optical sections which are ~ 0.75 micrometer thick. Importantly, confocal microscopy rejects light from out-of-focus planes, producing images which are remarkably detailed. Confocal microscopy can basically be thought of as a CAT scanner for cells. Confocal microscopy is being used to quantify probe fluorescence within specific subcellular compartments. In addition, it is possible to combine confocal and MDVM. With confocal MDVM, fluorescence can be determined for each compartment independently, allowing measurement of parameters within specific compartments even with probes which have little or no compartmental specificity. The confocal microscopes available in our facility provide both visible and UV wavelengths of excitation, and are capable of repetitively scanning a single line across an object in the image field at intervals between 10-100 msec. Should higher temporal resolution be required, the scanning component of the confocal can be deactivated and repetitive measurement can be made from the same spot repeatedly.
Confocal microscopy has been used by a number of investigators to localize specific cellular or tissue constituents in 3-dimensions in a variety of biological studies. These include localization of focal adhesion constituents in locomoting cells, the organization of various proteins comprising cell-cell junctions and spatial interrelationships between cellular organelles. A more recent emphasis has been on the use of the confocal microscope as an analytical tool to study the structure and physiology of living cells. Such measurements include cell volume and surface area, visualization of various organelles in living cells, mitochondrial and plasma membrane potential, mitochondrial and cytoplasmic pH and cytoplasmic, mitochondrial and nuclear calcium. These measurements have been applied to the studies of hypoxic, ischemic and toxic injury as well as growth factor signal transduction and cancer metastasis.
The C&DB Imaging Facility currently houses three laser scanning confocal microscopes. The Zeiss 410 has visible (488, 547, 568, and 647nm) and UV (350 and 364nm) lasers for excitation and is equipped with a temperature controlled stage. The Bio-Rad MRC600 (488 and 568nm excitation) also has a temperature controlled stage. The Zeiss 510 NLO is mated with our pulsed Ti-Sapphire laser to form our multi-photon excitation instrument. The Zeiss 510 also provides us with state of the art confocal imaging with five visible laser lines (458, 488, 514, 543, 633nm). In addition to temperature control the 510 has humidity and carbon dioxide regulation. To sign up for these instruments contact the C&DB personnel.
Data Examples
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7 day cultured Neonatal Cardiac Myocytes labeled with calein AM. The first image in the left column shows the outline of the myocyte that will be bleached. The second image is taken 4ms after a 3 second bleach and the third image is taken 7.5 min after the bleach. The right hand column is a group of cells treated with the gap junction inhibitor carbenoxolone. The images show that by blocking the gap junctions recovery is inhibited.
Fluorescence can be used as a spectroscopic ruler to study and quantify the interactions of cellular components on the molecular level. Fluorescence resonance energy transfer is a process by which a fluorophore (donor) in an excited state may transfer its excitation energy to a neighboring chromophore (acceptor) nonradiatively through dipole-dipole interactions. The usefulness of this technique derives from the fact that the efficiency of the energy transfer process varies as the inverse of the sixth power of the distance separating the donor and acceptor fluorophores, resulting in the ability to measure interactions between cellular components on a scale of 10-50Å . One of the cellular components to be studied is labeled with donor fluorophores and the other cellular component is labeled with acceptor fluorophores. By design, the emission spectrum of the donor fluorophore overlaps the excitation spectrum of the acceptor fluorophore. When the distance separating the donors and acceptors is less than or equal to 50Å, then energy from the excited donors can non-radiatively transfer to the acceptors. The extent of transfer is related to the distance separating the donor and acceptor fluorophores (and by design, the structures to which they are attached).
Lateral segregation of plasma membrane lipids during cell injury is being examined using FRETM. Measurements made using FRETM have provided a potential mechanism to account for the alterations in plasma membrane lipid order and topography attendant with hypoxic injury. A picture has emerged where hypoxia induces the formation of domains composed of identical phospholipids, and these domains become packed tighter and tighter as injury progresses, eventually causing holes or gaps in the membrane to occur leading to loss of the plasma membrane permeability barrier and the onset of irreversible injury. FRET is also being applied to test the hypothesis that growth factor binding results in receptor dimerization which is required for biological activity. These studies are focused on Platelet-derived Growth Factor receptors, as these receptors and their ligands play a major role in tumors of mesenchymal origin and atherosclerosis. The application of green fluorescent proteins (GFP) as donors and/or acceptors is also being explored in this model system. Lastly, FRETM is being employed to study the interaction between high risk HPV E6 protein and the tumor suppressor protein p53. FRETM demonstrated that HVP E6 and p53 were separated by a distance less than 50 Angstroms, and hence are most probably bound together in a complex. This study substantiated in vitro studies showing that E6 can bind p53 protein and indicate that part of the mechanism by which HPV-16/18 could cause cervical neoplasia may be that cytoplasmic HPV E6 binds to p53 protein after its transcription in the cytoplasm, preventing p53 from entering the nucleus where it performs its normal growth (tumor) suppressor function.
Two-Photon fluorescence excitation occurs by the simultaneous absorption of two photons each having half the energy needed for excitation. Efficient two-photon excitation requires a high spatial and temporal concentration of photons. This technique results in a image which is a thin slice of the specimen (as in confocal microscopy) but where the excitation is confined to the plane of focus. Consequently, photo-bleaching and photo-toxicity are reduced enabling longer observation times for live cell studies. Another advantage of this method is deeper penetration of the exciting light into scattering tissue as a result of the use of longer wavelengths.
In confocal microscopy, the pinhole is normally centered to the excitation spot to maximize the collection of light and to maximize the confocal effect. If the pinhole is shifted in the direction opposite to the direction of the scanning beam, delayed luminescence is selectively transmitted through the pinhole. This effect allows the emission from long-lifetime luminescent probes to be isolated from the signal from luminescent probes of shorter lifetime because as the pinhole displacement from center is increased the signal from a short-lifetime probe will be attenuated much faster than that from a long-lifetime probe. In the pair of images above the upper image was acquired with the pinhole centered and all the beads are visible: the two dim, long-lifetime beads on the right and the two brighter, short lifetime beads on the left. In the lower image the pinhole is shifted in the direction opposite the scan direction and the two long-lifetime beads remain visible, while the two short lifetime beads have disappeared. This technique will be used to visualize a long lifetime probe in a tissue which has sufficient short lifetime autofluorescence to obscure the long lifetime signal.
The principle of confocal imaging is the same in spinning disk confocal as in laser scanning confocal. However in laser scanning confocal a laser is required for the excitation source and the scanning beam excites only one spot in the specimen at each time. The spinning disk confocal may use a laser or an arc lamp excitation source and provides excitation of many spots simultaneously. The use of an arc lamp, as our spinning disk confocal does, allows the excitation wavelength to be changed by simply changing the excitation filter and wavelengths may be chosen that are not available from practical lasers. Thus calcium ion imaging may be accomplished with fura-2, the optimal calcium indicator that requires two UV wavelengths for ratio imaging. The excitation of many spots simultaneously permits the acquistion of the image by a video camera (instead of a photomultiplier tube) and monitoring biological processes at video rate (30 frames per second) is possible. The image above is from a three-dimensional reconstruction of some fluorescent beads. The original image stack was acquired with our Atto Instruments spinning disk confocal microscope and each of the original images represents a thin slice of the specimen. The image above is a view of the reconstruction at 45 degrees to the optical axis. If you would like to see views of the reconstruction from all angles