Some optical arrangements allow the observation of unstained cells and tissue sections. Unstained biological specimens are usually transparent and difficult to view in detail, since all parts of the specimen have almost the same optical density. Phase-contrast microscopy, however, uses a lens system that produces visible images from transparent objects (Figure 1–3).
Cultured neural crest cells seen with different optical techniques. The cells are unstained, and the same cells appear in all photographs. Two pigmented cells are used for orientation in each image. A: Conventional light microscopy. B: Phase-contrast microscopy. C: Nomarski differential interference microscopy. High magnification. (Courtesy of S Rogers.)
Phase-contrast microscopy is based on the principle that light changes speed when passing through cellular and extracellular structures with different refractive indices. These changes are used by the phase-contrast system to cause the structures to appear lighter or darker relative to each other, which makes this kind of microscopy a powerful tool with which to observe living cells. Another way to observe unstained cells or tissue sections is Nomarski differential interference microscopy, which produces an apparently three-dimensional image (Figure 1–3).
Polarizing microscopy allows structures made of highly organized molecules to be recognized. When normal light passes through a polarizing filter (such as a Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through. If, however, tissue structures containing oriented molecules (such as cellulose, collagen, microtubules, and microfilaments) are located between the two polarizing filters, their repetitive, oriented molecular structure rotates the axis of the light emerging from the polarizer. Consequently, they appear as bright structures against a dark background (Figure 1–4). The ability to rotate the direction of vibration of polarized light is called birefringence and is a feature of crystalline substances or substances containing highly oriented molecules.
Polarized light microscopy. A small piece of rat mesentery was stained with the picrosirius method, which stains collagen fibers. The mesentery was then placed on the slide and observed by transparency. Under polarized light, collagen fibers exhibit intense birefringence and appear brilliant or yellow. Medium magnification.
The depth of focus in the regular light microscope is relatively long, especially when small magnification objectives are used. This means that a rather wide extent of the specimen is seen in focus simultaneously, causing superimposition of the image of a three-dimensional object. With confocal microscopy, on the other hand, only a very thin plane of the specimen is seen in focus at one time. There are two principles on which this is based: (1) the specimen is illuminated by a very small beam of light (whereas in the common light microscope, a large beam of light floods the specimen) and (2) the image collected from the specimen must pass through a small pinhole. The result is that only the image originating from the focused plane reaches the detector whereas the images in front of and behind this plane are blocked (Figure 1–5). The harmful glare of the out-of-focus objects is lost, and the definition of the focused object becomes better and allows the localization of any specimen component with much greater precision than in the common light microscope.
Principle of confocal microscopy. While a very small spot of light originating from one plane of the section crosses the pinhole and reaches the detector, rays originating from other planes are blocked by the plate. Thus, only one very thin plane of the specimen is focused at a time.
For practical reasons, the following arrangement is used in most confocal microscopes (Figure 1–6): (1) the illumination is provided by a laser source; (2) because it is a very small point, it must be moved over the specimen (scanned) to allow the observation of a larger area of the specimen; (3) the component of the specimen that is of interest must be labeled with a fluorescent molecule (meaning that a routine section cannot be studied); (4) the light that is reflected by the specimen is used to form an image; (5) because the reflected light is captured by a detector, the signal can be electronically enhanced to be seen in a monitor.
Practical arrangement of a confocal microscope. Light from a laser source hits the specimen and is reflected. A beam splitter directs the reflected light to a pinhole and a detector. Light from components of the specimen that are above or below the focused plane are blocked by the plate. The laser scans the specimen so that a larger area of the specimen can be observed.
Because only a very thin focal plane (also called an optical section) is focused at a time, it is possible to reunite several focused planes of one specimen and reconstruct them into a three-dimensional image. To accomplish the reconstruction and many of its other features, the confocal microscope depends on heavy computing capacity.