Science begins with observation. The more accurate and precise the observation, the sounder the hypothesis, the more discerning the experiment, the more telling and reliable the result. Techniques that allowed us to observe organelles and molecules made possible a revolution in science resulting in the fields of cellular and molecular biology; these disciplines have laid the foundations of an impressive number of advances in many areas of biology, including the medical arena. The introduction of new optical imaging techniques, such as laser scanning confocal microscopy, has generated widespread excitement among biologists and is leading to a whole new generation of new discoveries.

Traditional imaging technologies, including light and electron microscopy, opened a window on the inner workings of cells and organisms, thus enhancing our understanding of the processes of life. Unfortunately, to see the interior of a specimen at high resolution using traditional microscopy, you typically have to slice the specimen into thin sections, interrupting 3-D relationships; this approach also necessitates prior killing and preservation of the specimen. It is this limitation that can be overcome by confocal microscopy. The confocal microscope opens a door that allows us to venture undisturbed into the three dimensional interior of cells, tissues, and even whole organisms.

A confocal microscope is a tremendous advance in microscopy that uses laser technology to optimize light microscope optics, pushing toward the resolution limit of the objective used (under favorable conditions, eg, small pinhole and bright specimen label, resolution can be improved as much as 1.4X over conventional fluorescence microscopy). In the last few years, it has become a very powerful analytical tool in cell and molecular biology, yielding spatial resolution of fluorescent labeled structures at the macromolecular level. In traditional epifluorescence microscopy, the whole specimen is bathed in a short wavelength light; typically the light has passed through a chromatic reflector which directs the short wavelength illumination onto the specimen. The emitted, longer wavelength light (from fluorescent labels within the specimen) passes straight through the chromatic reflector into the eyepiece.

The principle of confocal microscopy is to illuminate the specimen with a laser light focused at one position in the specimen, using only a single point of illumination. This illuminated point is swept back and forth in the X and Y directions across a Z-plane the specimen, then refocused at a different depth (Z), swept, refocused, swept, and so on until the specimen has been viewed at all layers; the process is called optical sectioning to contrast it with a physical sectioning using some kind of a knife. The laser excites a fluorescent label, and the light emitted is picked up by a detector and converted to an electrical signal fed to the computer. The illuminating beam passes first through a pinhole so that a diffraction limited spot is focused by the objective onto the object; this means that the illumination intensity immediately above and below the focal plane is quite reduced due to beam convergence and divergence. Thus there is decreased emission of fluorescence from outside the focal plane. Light emitted from the excited fluorochrome of the specimen within the illuminated volume then passes through a dichroic reflector and through a pinhole conjugate to the illuminating pinhole.

Light scattered from parts other than the point of specimen illuminated is rejected from the optical system [by the exit pinhole] to an extent never before realized (Minsky, 1988)

One of the limits faced by the microscopist has to do with the size of the pinhole forming the illuminating spot. The size of this aperture is adjustable; up to a point, the smaller the hole, the better the resolution. However, there is a tradeoff here, for as the hole gets smaller, the illuminating intensity diminishes and the resuting emitted signals likewise are diminished, necessitating increased amplification and its accompanying noise. There is a limit to the amount of light that can be emitted from the amount of fluorochrome found within the illuminated volume. Excess excitation causes all the fluorochrome to be in the excited state - and this state has a finite lifetime; this results in a saturation of emission where more illumination does not result in more emission, only damage to the specimen. An additional parameter is the speed of the scan; too fast and there is minimal emission, while too slow results in overexcitation.

The signals collected by the photomultipliers of the confocal microscope detector are assembled in digital form and stored by a computer. The computercan then carry out a variety of digital image processing techniques, including brighttness and contrast manipulations, pseudocoloring, edge enhancements (sharpening or smoothing), etc. Perhaps most impressive is the reassembly of the optical slices into a three dimensional picture of the specimen. The specimen itself can stay completely intact, reducing the distortion that can accompany physical sectioning and obviating the need for guesses about the relationships between structures. Since the emission is very tightly localized to the fluorochrome, the image quality (resolution) is greatly enhanced by a reduction of background scatter. This allows for accurate morphological analysis. The ability to image, in focus, a Z-series of micrographs gives the investigator the ability to optically section a specimen of interest and to create three-dimensional reconstructions, an impossibility with conventional fluorescence microscopy. With appropriate computers and advanced image processing and analysis software, multiple recorded optical slices can he superimposed, giving an extended focus image which can only be achieved in conventional microscopy by reduction of the aperture and thus sacrificing resolution. Any part of the reassembled three dimensional image can be viewed in context and from any angle. Thus the user gets multidimensional data - spatial and spectral. Even more remarkably, under the right circumstances it is possible to observe the microscopic components of cells and organisms that are still alive. Thus there is temporal information as well, meaning that with the confocal microscope one can measure dynamic events within the complex sub-cellular environment of a living specimen.

The Leica microscope also has the capability for DIC (Nomarski) imaging, which is a modification of phase contrast microscopy. In the latter, the image formed is the result of refractive index (RI) differences in the sample, as opposed to light absorption differences in traditional brightfield microscopy. When light passes through areas of differing refractive indexes, the phase of the light is shifted - put out of step with waves that have passed through areas of a different RI (light travels slower through materials of a higher RI). Contrast is generated by converting these phase differences back into amplitude differences; in other words changes in light intensity reflecting differences in RI within the specim3en as compared to the background. The main drawback is that every object is surrounded by a diffuse halo of light; this cannot be removed. Nevertheless, phase contrast microscopy is extremely valuable in looking at unfixed, unstained, or living objects, as the different RI's are responsible for generating the contrast.

However, Differential Interference Contrast Microscopy is a different phase imaging technique that can eliminate these phase rings in transparent objects. It uses plane polarized light which then passes through a beam splitter in the condenser resulting in two beams of light whicha re laterally displaced wiuth respect to eachother (shear) and polarized at right angles to eachother. These pass through the specimen at differnt places, then into the ojective and beam combiner to be recombned into a single beam - thus producing contrast differences. Edges are interestng here, for one side appears brighter, the other darker, giving the impression of shadowing or "3D". However, these edges are sharp; they do not present the halo effect of phase microscopy.