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 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% (Figure 2-6A). The condenser slit is adjusted so that light transmission through the slit falls on the gray region (15%) of the modulator.
The HMC microscope exploits phase gradients within the sample. In regions where there is a rapid spatial change of sample optical path, refraction will occur, slightly shifting the path refracted light.17 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 will experience no refraction and pass through the central gray region of the modulator and be rendered uniformly 15% gray. Light passing through positive gradients (dark to light) will be refracted through the light zone and remain bright. 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. Furthermore, the two polarizing filters (P1, P2) are placed optically in front of the sample. Thus, HMC can be used with birefringent specimens not amenable to DIC (e.g., crystalline objects or specimens in plastic Petri plates).
The only adjustment required for a microscope equipped with Hoffman modulation contrast is to align the slit condenser filter with the orientation of the modulator in the objective. Install a Bertrand lens or phase telescope in position and focus on the modulator as well as the slit filter. Then rotate and/or translate the filter until the slit edges align with the modulator gray-area edges.
17A plane wavefront passing through a negative or positive phase gradient will be diffracted obliquely, relative to the optical axis, an amount proportional to the gradient of optical path difference within the specimen. These spatially shifted wavefronts will be transmitted through either the light or dark areas of the Hoffman modulator.