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The Principles of Modulation Contrast

A Step by Step Guide

Making unstained live specimens visible is a major problem in microscopy. Depending on the specimen, the application and even on the coverslip, dish or immersion medium, several so-called contrasting methods have been developed to overcome this problem.

One contrasting method especially suited for use with plastic dishes and live cells is modulation contrast. Originally invented by Robert Hoffman in 1975, the modulation contrast converts phase gradients inherited in the sample structure into differences in brightness by employing oblique illumination. Modulation contrast creates 3D-like images of unstained specimens, rendering it the contrasting method of choice for applications like in vitro fertilization, where DIC is not possible (due to the use of plastics) or phase contrast does not deliver satisfying results.

This tutorial explains the optical elements in the light path and the operating mode of modulation contrast, taking the example of an an inverted and motorized high-end research light microscope which can be used for transmitted light contrasting methods and fluorescence microscopy.

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