Phase contrast microscopy made it possible to observe living cells.

Traditional bright-field micro­scopy gives low contrast images of unstained cells (left). Phase contrast microscopy (right) enhances the contrast.

At the beginning of the 20th century the image quality of the optical microscope had reached the quality of modern optical microscopes. However, it was still difficult to obtain high contrast images of living cells.

In 1932 Frits Zernike approached Carl Zeiss AG,the leading microscope manufacturer at the time, with a new type of microscope which improved the contrast of living cells. Zernike's own words from his Nobel Lecture (Zernike 1953) — I went in 1932 to the Zeiss Works in Jena to demon­strate. It was not received with such enthusiasm as I had expected. Worst of all was one of the oldest scientific associates, who said: «If this had any practical value, we would ourselves have invented it long ago».

Zeiss did eventually build a microscope based on Zernike's invention in 1936. His inven­tion, the phase contrast microscope, is today an everyday tool for all cell biologists. In 1943, the first time-lapse film imaged with the Zeiss phase contrast microscope was a sensation, showing cell division.

Two years after Zernike received his Nobel price, in 1953, Georges Nomarski published the theory for the second popular phase contrast microscopy method —  differential interference contrast (DIC) microscopy. Just like Zernike's phase contrast, Nor­marski's phase contrast enhances the contrast of transparent objects. But, instead of high­lighting edges, the Normarski method achieves contrast by artificially creating shadows.

Conventional phase contrast microscopy creates visual artifacts to visualize unstained cells (left). Quantitative phase microscopy achieves contrast by quantifying variations in optical distance (right).

Quantitative phase contrast microscopy

Transparent objects are difficult to see because they do not absorb or scatter light. Light do, however, move slower when passing through a transparent object which is more optically dense than the surrounding. The illuminating microscopy light thus travels a longer optical distance when passing through a transparent object.

Both conventional phase contrast methods visualize such differences in optical distance. But, they do not quantify the difference. Quantitative phase contrast microscopy is made possible by the develop­ment of high resolution digital image sensors. By computer processing image information, quantitative phase contrast mea­sures differences in optical distance. These measurements can either be used to calculate the optical thickness of the object or to create a topographic image of the object.

Working principle of phase contrast microscopy

The basic principle to make phase changes visible in phase contrast microscopy is to separate the illuminating background light from the specimen scattered light, which make up the foreground details, and to manipulate these differently.

The ring shaped illuminating light (green) that passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The remaining light is unaffected by the specimen and form the background light (red). When observing unstained biological specimen, the scattered light is weak and typically phase shifted by -90° — relative to the background light. This leads to that the foreground (blue vector) and the background (red vector) nearly have the same intensity, resulting in a low image contrast (a).

In a phase contrast microscope, the image contrast is improved in two steps. The background light is phase shifted -90° by passing it through a phase shift ring. This eliminates the phase difference between the background and the scattered light, leading to an increased intensity difference between fore­ground and background (b). To further increase contrast, the background is dimmed by a gray filter ring (c). Some of the scattered light will be phase shifted and dimmed by the rings. However, the background light is affected to a much greater extent, which creates the phase contrast effect.

The above describes negative phase contrast. In its positive form, the back­ground light is instead phase shifted by +90°. The background light will thus be 180° out of phase relative to the scattered light. This results in that the scattered light will be subtracted from the background light in (b) to form an image where the foreground is darker than the background.

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