In regions of the sample where there is a rapid spatial change of sample optical path (say from the outside to the inside of a cell in culture), refraction will occur, slightly shifting the path impinging light. Light passing through negative amplitude gradients (light to dark) within the sample will be refracted through the dark zone of the modulator and be rendered darker. Light that passes through homogeneous areas within the sample (say the center of the cell) will experience no refraction and pass through the central gray region of the modulator and be rendered uniformly gray. Light passing through positive gradients (dark to light) will be refracted through the light zone and remain unchanged. Thus, light passing through the modulator is accentuated in contrast and results in an image with pseudo-relief (much like DIC) with the three-dimensionality representative of light phase gradients rather than actual object geometry. Unlike DIC, HMC uses no beam-splitting prisms, and no wave interference occurs. HMC can be used with birefringent specimens not amenable to DIC (e.g., crystalline objects or specimens in plastic Petri plates).

Hoffman modulation contrast (HMC)

The Hoffman modulation contrast (HMC) microscope is another method by which transparent, or nearly so, objects can be visualized. Hoffman modulation contrast accentuates phase gradients within the sample and displays them in the image plane as levels of gray modulated lighter and darker than an average background gray. To accomplish this, a special filter is placed in the Fourier plane (back focal plane) of the objective (Figure 2-6D) conjugate to another filter, the condenser slit. The modulator filter is constructed so as to have three regions of different neutral densities, ranging from low to a high attenuation, usually T=100%, 15%, and 1%. The condenser slit is adjusted so that light transmission through the slit falls on the gray region (15%) of the modulator. A small Polarizer filter in the Hoffman slit may be adjusted to modify the image contrast.