With transmitted light microscopy, images result from light (halogen lamp source) passing through the specimen. Specimen 'details' will be evident if the specimen and background differently alter the phase of light – giving contrast between the specimen and background. Since cells are made mostly of water (70%), they do not alter the passage of light enough to provide much of an image. Therefore, when imaging a specimen that is poorly visible, contrast may be increased by fixing and histologically staining the specimen, or by using microscope optics to accentuate the small differences in the phase of light that the specimen causes (ie DIC, phase contrast, dark-field).
Differential Interference Contrast (DIC)
With DIC microscopy, two slightly separate, plane polarized beams of light are used to create a 3D-like image of the unstained specimen. A polarizer, placed just after the light source, creates plane polarized light that is passed through a Wollaston prism which splits the beam into two beams that have their axis of vibration at 90° relative to each other. The beams cross and go through the condenser and emerge separated by a small distance. The two beams go through slightly different parts of the specimen where small differences in refractive indexes of the cellular components alter their wave path lengths differently. Next, the two beams enter the objective, and pass another Wollaston prism that recombines them. This second prism is offset slightly from the first, creating a difference between deviated and undeviated beams. Since the two beams went through slightly different parts of the specimen, they will have different path lengths; therefore the waves can interfere if they are vibrating in the same plane. Therefore, the combined beam passes a second polarizing filter that is 90 degrees to the first one, which polarizes the bipolar beam and so the combined polarized light can have negative interference – gives light and dark areas of specimen.
Phase contrast microscopy uses an annular stop in the condenser and a phase plate within the objective lens, which is aligned with the annular stop. In this configuration, the light path can be split and each of the separated beams will pass through the specimen at the same place, as a cone of light at the focal point. Any background light, which is un-deviated by the specimen, will go through the phase ring in the phase plate and is advanced by a quarter of a wavelength. Deviated light passing through the specimen is retarded by a quarter of a wavelength and passes through the phase plate without going through the ring. When the beams are recombined further along the light path, the differences in the phase of the deviated and un-deviated light beams become additive and subtractive. The resultant wave is the sum of the two waves which have their crests and troughs opposite each other. The difference in amplitude can be seen as a change in brightness, since brightness is proportional to the square of the amplitude. The net result is that features of the object are either lighter or darker than the surrounding field.
With Dark-Field microscopy, oblique light rays illuminate the specimen, and are not transmitted directly through the objective. Only light rays that are diffracted by the specimen into the objective aperture are collected resulting in a dark background and a bright specimen.