From Ruzin, 1999. Plant Microtechnique and Microscopy
Phase contrast microscopy imparts contrast to unstained biological material by transforming phase differences of light caused by differences in refractive index between cellular components into differences in amplitude of light, i.e., light and dark areas, which can be observed. As light rays pass through areas within the tissue of different optical path (refractive index and geometric path length) they may be retarded in phase by up to 1⁄4Ü but will remain unchanged in amplitude. Since the eye cannot discern phase differences, a mechanism for transforming phase changes into amplitude changes is required.
In the early 1950s Zernike13 discovered the method by which phase differences can be transformed into amplitude differences. Zernike invented what is now known as positive or dark phase contrast. An alternate method, negative or bright phase contrast, was subsequently developed and has supplanted Zernike’s original approach. In positive phase contrast the object (e.g., cell component) appears darker than the surrounding background. In negative phase contrast the object appears brighter than the background.
13Frits Zernike (1888–1966) received a Nobel prize in 1953 for his discovery of phase contrast.
A compound microscope equipped for negative phase contrast has two additional components: a “phase plate” that retards light exactly 1⁄4 wavelengthin a centered, ring-shaped area located at the back focal plane of the objective lens and a matching “phase annulus” in the condenser consisting of a clear ring on a black field (Figure 2-3B). The presence of the annulus and matching phase plate causes the direct (unmodified background) light to pass only through the phase ring and thus be retarded 1⁄4Ü. Because the light intensity of the diffracted light will be slightly diminished by absorption within the specimen, a neutral density coating on top of the phase ring attenuates undiffracted, background light to balance total illumination.
The light rays interacting with the specimen, on the other hand, diffract away from the sample as spherical waves that do not impinge on the phasing areas of the phase plate to any appreciable degree, but are focused by the objective onto the image plane. Due to a difference in optical path
between the specimen and the surrounding medium, the refracted waves will be retarded in phase up to 1/4 wavelength.
Thus, in the correctly adjusted phase contrast microscope, there are two possible light paths. Light that does not interact with the specimen is collected by the objective, passes through the phase plate ring, and is retarded exactly 1⁄4 wavelength. The phase shift is not detectable by the eye so the resulting image on the image plane in the microscope appears as a normal bright background.
Conversely, light that passes through the specimen may be diffracted by edges and local irregularities within the tissue. This refracted light will be retarded in phase by up to 1⁄4Ü. The diffracted light will diverge from the object, fill the back focal plane of the objective, and be resolved on the image plane within the microscope. Since the background light is restricted to, and attenuated by, the relatively small annular phase plate, the light diffracted by the specimen can assume a significant role in image construction at the image plane.
Light from both possible paths (background path and specimen path) will interact at the image plane resulting in wave interference where light from the specimen interacts with light from the objective phase plate. In negative phase contrast, constructive interference occurs at the image plane. The results of this interference are bright areas in the specimen image that correspond to refractive index differences in the specimen itself (organelles, cell walls, etc.) set against a background of “normal” intensity derived from nonrefracted light.
Phase contrast objects always have a “halo” of light (either bright around dark objects or dark surrounding bright objects), which is the result of diffracted light passing through the phase ring as well as the nonphase areas and interacting at the image plane. This halo, also referred to as shading-off, is representative only of light diffraction and interference and not of the optical path of the sample itself. That is, the halo adds artificial structure to the specimen. Intensities seen at the image plane are the result of optical path difference (refractive index plus geometric distance) within the specimen and may not necessarily represent the actual structure of the specimen.
In practice adjustment of the microscope for phase contrast is simple. Objectives equipped with a phase plate are coded “Ph1,” “Ph2,” or “Ph3” depending on the lens magnification and the size of the phase rings. Phase objectives must be matched to the appropriately sized annular diaphragm in the condenser by rotating the condenser to the Ph1, Ph2, or Ph3 position. By placing a lens (Amici-Bertrand lens or ‘phase telescope’) that focuses on the back focal plane of the objective into
14A vector diagram helps explain the phase relationships that exist in phase contrast microscopy. Amplitude is represented by the length and phase by the angle of the arrow.
The light diffracted by a nonabsorbing microscopic object with a higher refractive index than the surrounding medium will be retarded in phase usually only a small amount. The phased light (p) may be treated as the summation of incident light (u) plus a new vector (d) that has a higher phase shift. With most biological material, d may be shifted in phase up to ±1⁄4Ü.
Under normal circumstances u and p are indistinguishable at the image plane because their amplitudes are equal (or nearly so) and thus a phase object is invisible. However, by artificially advancing (A; u1) or retarding (B; u2) background light 1⁄4Ü (±90°), a vector can be created from the sum of u1 or u2 plus the diffracted light vector d. This new vector is either shorter (p1) or longer (p2) than the background light vector (u1 or u2) and thus is visible as either a dark or light spot (respectively) on the image plane.
Vector diagram of the phase relationships between background and object light in a phase contrast microscope.