Brief History:

In the history of microscopy, one of the challenges microscopists faced was producing enough contrast when observing thin tissue sections and living organisms without needing to kill, fix, dehydrate and then stain the specimens with dyes. Other microscopy techniques like dark-field, oblique (relief), polarizing, and Rheinberg lighting were also developed and are easy to implement. In the 1930s a Dutch physicist by the name of Fritz Zernike developed a new technique called phase-contrast that was so significant that he garnered the Nobel Prize in 1953.

Our eyes are sensitive to changes in movement, wavelength (colour), and amplitude (brightness) but not to changes in phase. A phase contrast microscope converts changes in phase to changes in amplitude which our eyes can see. Most living cells and many aquatic microorganisms appear like translucent “bags of water” when viewed by bright light microscopy. There are some dyes that in low concentration can be used to stain living cells (e.g. iodine, acridine orange, methylene blue, Nile red) but most are toxic at high concentrations and the dyes can alter an organism or cells behaviour.  Phase contrast is a technique that can be used to view living cells or organisms with a minimum of deleterious effects. One other advantage of phase contrast microscopy is that it is insensitive to polarized light and birefringence and can be used to observe cells cultured in plastic dishes.

BASIC PHASE CONTRAST DESIGN

A phase contrast microscope uses several optical techniques to produce contrast within living cells. The first is a circular annulus in or below the condenser that provides a cone of partially coherent light focused onto the specimen. The light passes around and through the specimen. Some of this light is diffracted as it passes through the specimen and the light waves are retarded by ¼ of wavelength on average. This diffracted light and the direct unaffected light passing around the specimen enters the objective. Both the direct light and some of the diffracted light passes through a phase ring at the back of the objective.

The phase ring has two functions 1) it reduces the overall brightness of the direct light, so it doesn’t overwhelm the diffracted light and 2) it slows (retards) or speeds up (accelerates) the direct light by ¼ wavelength so that the direct light and diffracted light are ½ a wavelength out of phase causing constructive or destructive interference. Constructive interference makes cellular components brighter (negative phase contrast) and destructive interference makes them darker (positive phase contrast). The phase ring is also grey, and you can see it by looking through the back of the objective. The resultant changes in amplitude caused by interference between the direct and indirect (diffracted) light become visible as differences in brightness and contrast when living organisms are viewed by phase contrast.

The annular diaphragm creates a cone of light focused on the specimen. The specimen causes some of the light to be become diffracted and the light is retarded in phase by ¼ of a wavelength. Direct light proceeds to the objective and around the specimen and some of it passes through the phase plate rings where it is attenuated, and the phase altered by ¼ wavelength.  The direct light and diffracted light interfere resulting in interference causing changes in brightness and contrast.

PHASE ANNULUS (ANULAR DIAPHRAGM)

The Phase Annulus is a black disk with a clear ring or slit that sits in or under the condenser. Its’ purpose is to provide a cone of light that is focused on the specimen. Unlike dark field lighting, light from the phase annulus enters the objective. Different objectives require different sized annuli that match the objectives (in practice one disc may support more than one objective e.g. Motic Phase 2 disc supports both the 20X and 40X objectives). Zernike experimented with different slit patterns (see Fig. 7 Pelc et. al. 2020) but the circular disc is the most common one in use today in part because it was the easiest to align with the phase plate such that halo artifacts are spread in an angular direction.

The function of the phase annulus is also to provide partially coherent light. Coherence occurs when the majority of light of a single color is in phase – i.e. most of the light waves are in sync. Laser light is perfectly coherent but it turns out it is too coherent (Hard et. al. 1972) and creates optical noise by revealing flaws and dust in the optical system. The other function of the phase annulus is to match the phase disc at the back of the objective focal plane so the direct light can be directed and aligned with the phase plate.

PHASE PLATE

The phase plate is a circular disk usually positioned at the back of the objective focal plane. The disc must be perfectly aligned with the phase annulus in the condenser. It serves two purposes: the first is to attenuate the direct light by about 75% to match the relative intensity of the diffracted light. The attenuation of the direct light in the past was done using a thin layer of soot, today the rings are coated with thin metallic films. The second function of the phase ring is to change the phase of the direct light by a ¼ wave relative to the diffracted light. If the direct light is retarded ¼ of a wave it then comes in phase with the diffracted light resulting in constructive interference (negative phase) or it advances the phase of the direct wave by ¼ wavelength so that the direct and diffracted light waves are now ½ wave out of phase resulting in destructive interference (positive phase contrast).

Positive phase contrast is the most common type found in light microscopes.  With positive phase contrast, thicker organelles in the cell appear dark against a light background e.g. nucleus and mitochondria. Positive phase contrast is commonly used for examining cells in culture or aquatic microorganisms.

Negative phase contrast makes thicker regions of the cell and organelles appear bright against a dark background. The direct light is retarded by ¼ wave producing constructive interference resulting in bright details on a dark background. Negative phase is good for observing some protists e.g. vorticella. Nuclei in cheek cells appear bright white by negative phase contrast.

“Motic offers both positive and negative phase objectives”

Some microscopes have the phase plates positioned outside of the objectives such that they can use ordinary bright field objectives to produce phase contrast (e.g. some inverted microscopes). Motic also offers inverted phase contrast microscopes.

ADDING PHASE CONTRAST TO AN EXISTING SCOPE

Adding phase contrast to an existing microscope is one of the best ways to upgrade a light microscope. It is important to note that while some phase condensers and phase objectives from different microscope manufacturers may work together many will not because the phase rings won’t align with the condenser annuli.  The cost of a phase-contrast system varies with the microscope brand, the quality of the objectives purchased (Achromat, Plan Achromat, Fluorite, Apochromat), and type of phase contrast. Adding phase contrast to a microscope is significantly lower in cost than adding fluorescence or differential interference contrast (DIC).

Phase-contrast microscopy does have some small disadvantages. The first is that cells often have bright or dark halos around them and their intracellular components that can reduce the resolution, but these halos also serve to enhance contrast. The halos can be reduced or eliminated in several ways including computer processing, altering the refractive index of the medium, and using special apodized phase rings in the objectives that reduce edge effects (Pelc et. al. 2020) Using a 100X phase objective requires a reasonably bright light source and oil immersion fluid.  Finally, thick specimens can sometimes be difficult to interpret in phase contrast.

Posted by Motic America on February 16, 2021