Phase contrast microscopy explained

Phase contrast microscopy is an effective and convenient method of boosting contrast, revealing hidden details in transparent and unstained specimens.

It was invented by Dutch physicist Frits Zernike in the 1930s, who was awarded the Nobel prize for his discovery.

Phase contrast works by converting diffracted light into visually brighter or darker areas in the microscope image. This approach allows you to see greater detail and previously invisible structures.

Easily incorporated into most microscopes, it can also be combined with fluorescence and other techniques for more detailed visualisation.

How does it work?

Phase contrast microscopy works by converting differences in light phase (which we can’t normally see) into differences in brightness (which we can see).

When light passes through a specimen, it scatters (or diffracts) as it encounters denser areas. The light slows down as it diffracts, leaving it slightly out of phase with the light that passes through without getting diffracted.

Phase contrast microscopes magnify this phase difference between the diffracted and undiffracted light, resulting in constructive or destructive interference when they overlap.

The interference results in brighter or darker regions, revealing hidden details and structures with now greater contrast.

Advantages

There are several advantages to phase contrast microscopy:

• It significantly increases contrast in transparent and colourless samples, such as cells and microorganisms, making previously invisible details such as internal structures or cilia easy to see.
• It’s a purely optical technique that does not require harmful stains, making it suitable for observing living organisms or cell cultures in their natural state.
• It can be used in combination with other techniques, such as fluorescence microscopy, to achieve even greater levels of detail or to offset undesirable side-effects such as photobleaching.
• It is relatively inexpensive compared to other contrast-enhancing microscopy techniques, although more expensive than standard brightfield microscopes.

Applications

Phase contrast microscopy is widely favoured in a variety of fields for its effectiveness, ease of use and affordability.

In the realm of biological and medical research, it is extensively used due to its capacity to observe living samples without causing harm, and with minimal preparation.   

It also finds applications in mineralogy, crystallography, polymer assessment and quality control for commercial products.

Phase contrast is quite easy to acquire – you can either purchase a phase contrast microscope outright or modify your existing brightfield microscope with a phase contrast kit.

These kits typically consist of phase contrast objectives, condenser annular plates and a centering telescope.

Phase contrast is a bit more expensive than a standard brightfield microscope, but the amazing contrast and the ability to observe living specimens more than make up for the price difference.

Learn more on How to align a phase contrast microscope annulus