The most fundamental distinction between differential interference contrast (DIC) and phase contrast microscopy is the optical basis upon which images are formed by the complementary techniques. Specimens examined by these contrast-enhancing methods produce images that are often quite different in appearance and character when objectively compared. This interactive tutorial explores many of the similarities and differences exhibited between images captured with phase contrast and DIC microscopy.
The tutorial initializes with a randomly chosen specimen appearing the Specimen Image window, and the Contrast Mechanism slider is also set to a random position. In order to operate the tutorial, use the mouse cursor to translate the Contrast Mechanism slider between the Phase Contrast Image (slider to the extreme left) and DIC Image (slider to the extreme right) positions. As the slider is moved from one contrast mechanism to the other, the image changes character and begins to acquire the attributes of the new contrast mechanism. A different specimen can be selected from the palette by using the Choose A Specimen pull-down menu.
Phase contrast microscopy produces image intensity (amplitude) values that vary as a function of specimen optical path length magnitude, with very dense regions (those having large path lengths) appearing darker than the background. Alternatively, specimen features that have relatively low thickness values, or a refractive index less than the surrounding medium, are rendered much lighter when superimposed on the standard (positive) phase contrast medium gray background.
The situation is quite dissimilar for differential interference contrast, where optical path length gradients (in effect, the rate of change in the direction of wavefront shear) are primarily responsible for contrast. Steep gradients in path length generate excellent contrast, and images display the pseudo three-dimensional relief shading, which is characteristic of the DIC technique. Regions having very shallow optical path slopes, such as those observed in extended, flat specimens, produce insignificant contrast and often appear in the image at the same intensity level as the background.
Aside from the differences in contrast-formation mechanisms, DIC and phase contrast images differ in a number of other features. Illustrated in Figure 1 are several digital images comparing specimens captured in DIC and phase contrast. A human buccal epithelial cell revealing the nucleus and numerous bacteria on the upper surface in DIC is presented in Figure 1(a), and the same viewfield with phase contrast in Figure 1(b). The phase contrast image features pronounced halos around the cellular periphery and nucleus, which are absent in the DIC image. Optical sectioning DIC investigations (not illustrated) reveal that the bacteria are present on the membrane surface that is bathed in surrounding media as opposed to lying on the underside of the cell. This fact cannot be unambiguously determined with phase contrast.
In Figure 1(c), a thick section of murine kidney tissue imaged with DIC shows a bundle of cells enclosed in a tubule. A phase contrast image (Figure 1(d)) of the same area is confusing and disturbed by the presence of phase halos outside the plane of focus. However, several of the cellular nuclei appear visible in the phase contrast image, which are not distinguishable in DIC. Relatively high magnification views of an Obelia polypoid annulated stem perisarc in DIC and phase contrast are illustrated in Figure 1(e) and 1(f), respectively. In DIC, (Figure 1(e)) the annular structure appears hemispherical with internal rays that emanate from the stem. In addition, granular particles are visible within the stem structure, but anatomical detail is largely undefined. The phase contrast image (Figure 1(f)) is confused by halos around the annular rings and within the stem.
A primary advantage of differential interference contrast over phase contrast is the ability to utilize the instrument at full numerical aperture without the masking effects of phase plates or condenser annuli, which severely restrict the size of condenser and objective apertures. The major benefit is improved axial resolution, particularly with respect to the ability of the DIC microscope to produce excellent high-resolution images at large aperture sizes.
Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.