The utilization of contrast enhancement techniques in optical microscopy affects the response when relative modulation is calculated as a function of specimen spatial frequency. This interactive tutorial explores the effects of popular contrast modes on image contrast and the modulation transfer function of the modified microscope.

To operate the tutorial, click on the checkboxes adjacent to each contrast enhancing technique name to activate display of a calculated modulation transfer function curve. When the microscope is operating in brightfield mode (termed incoherent illumination; the tutorial default) using the full numerical aperture of the objective and substage condenser, resolution reaches the maximum theoretical limits, but image contrast at high spatial frequencies is reduced. This curve is denoted (1) in the checkbox and also adjacent to the MTF plot in the graph.

Coherent illumination (2) involves closing the substage condenser aperture diaphragm to a minimum, thus severely reducing the effective numerical aperture. This curve was calculated by Dr. Gordon W. Ellis, utilizing the fractional area of the condenser aperture that should be transmitted by the objective lens for light rays diffracted by the specimen at angles determined by the specimen period. Contrast is very high at low spatial frequencies, but quickly falls to zero as the frequency rises above 2.5 cycles per micrometer.

In the case of oblique illumination (3), specimen contrast is produced by oblique light rays emanating from a narrow annular opening in the substage condenser. It is evident from the resulting MTF curve that image contrast is improved for regions of the specimen having features with high spatial frequency, but overall contrast is considerably reduced at lower spatial frequencies ranging between zero and two cycles per micrometer. In this situation, specimen contrast is also governed by the position and size of the annular opening with respect to the microscope optical axis.

When a circular annular ring is placed in the front focal plane of the substage condenser that is conjugate to an amplitude phase ring at the objective rear focal plane (typical of a microscope operating in phase contrast mode), the MTF curve illustrated in case (4) is obtained. As is evident in the curve, overall contrast is improved over brightfield illumination with peaks occurring at spatial frequencies of 1 and 2.75 cycles per micrometer. The size and shape of this curve can be varied by altering the size of the condenser annular ring and the diameter of the amplitude ring in the objective.

Differential interference contrast (DIC) illumination (5) produces an MTF curve somewhat similar to phase contrast (4), but that varies with the angle between the period in the specimen and the shear direction of the Wollaston or Nomarski prisms. At low spatial frequencies, specimen contrast is lower than with brightfield illumination, but rapidly increases and reaches a peak around 1.5 cycles per micrometer before slowly declining once again.

Single sideband edge enhancement microscopy (6) produces superior contrast at high spatial frequencies with a good overall contrast response throughout the specimen period range. From the results described above, it is clear that contrast-enhancing techniques, such as phase contrast, DIC, single sideband edge enhancement, and oblique illumination produce images having more contrast at higher spatial frequencies.

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