33.2:
Phase Contrast and Differential Interference Contrast Microscopy
Unstained living cells are transparent and hard to see under an optical microscope. Phase-contrast and differential-interference contrast or DIC microscopes allow their visualization by creating higher resolution and sharper contrast images.
When a sample is illuminated in-phase-contrast, some light is diffracted by the cell components, while the rest directly passes through. The extent of diffraction depends on the refractive indices of the cell components.
As the diffracted and the direct light interfere, parts of the sample appear bright if the waves are in-phase and dark if they are out-of-phase. This creates high-contrast images of large organelles and thin tissue sections.
Sharp changes in refractive indices produce a halo around the image.
Such artifacts are absent in DIC, which uses a polarized light source to modulate the light waves passing through the sample.
The image contrast depends on the rate of change of the refractive indices across the specimen. Steep changes generate high-contrast images with an apparent three-dimensional effect.
DIC helps visualize thick specimens like brain slices, eggs, or embryos.
33.2:
Phase Contrast and Differential Interference Contrast Microscopy
Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of light focused on the specimen before reaching the objective lens. The objective contains a phase plate with a phase ring. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate.
Because waves have peaks and troughs, they can add together (if they are in-phase) or cancel each other out (if out-of-phase). When the wavelengths are out-of-phase, wave troughs cancel out wave peaks, which is called destructive interference. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness. As it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Specific cellular structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are well visualized with phase-contrast microscopy.
Differential Interference Contrast Microscopes
Differential interference contrast (DIC) microscopes (also known as Nomarski optics) are similar to phase-contrast microscopes in that they use interference patterns to enhance the contrast between different features of a specimen. In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. Once the beams pass through either the specimen or specimen-free space, they are recombined. The effects of the specimens cause differences in the interference patterns generated by combining the beams. This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens.
Suggested Reading
- Parker, N. et al. Microbiology: OpenStax., 2022. Section 2.4
- Frohlich, V.C., 2008. Phase contrast and differential interference contrast (DIC) microscopy. JoVE (Journal of Visualized Experiments), (18), p.e844.
- Palima, D. and Glückstad, J., 2010. Generalised phase contrast: microscopy, manipulation and more. Contemporary physics, 51(3), pp.249-265.
- Obara, B., Roberts, M.A., Armitage, J.P. and Grau, V., 2013. Bacterial cell identification in differential interference contrast microscopy images. BMC bioinformatics, 14(1), pp.1-13.