Understanding Bright‐Field and Phase‐Contrast Microscopy.

Different types of light microscopes use different types of illumination.

In a bright‐field microscope, the cone of light that illuminates the specimen is seen as a bright background against which the image of the specimen must be contrasted.

Bright‐field microscopy is ideally suited for specimens of high contrast, such as stained sections of tissues, but it may not provide optimal visibility for other specimens.

We will consider various means of making specimens more visible in a light microscope.

Bright‐Field Light Microscopy

Specimens to be observed with the light microscope are broadly divided into two categories:

A whole mount is an intact object, either living or dead, that can consist of an entire microscopic organism such as a protozoan or a small part of a larger organism.

Most tissues of plants and animals are much too opaque for microscopic analysis unless examined as a very thin slice, or section.

To prepare a section, the cells are first killed by immersing the tissue in a chemical solution, called a fixative.

A good fixative rapidly penetrates the cell membrane and immobilizes all of its macromolecular material so that the structure of the cell is maintained as close as possible to that of the living state.

The most common fixatives for the light microscope are solutions of;

formaldehyde,
alcohol,
or acetic acid.

After fixation, the tissue is dehydrated by transfer through a series of alcohols and then usually embedded in paraffin (wax), which provides mechanical support during sectioning.

Paraffin is used as an embedding medium because it is readily dissolved by organic solvents.

Slides containing adherent paraffin sections are immersed in toluene, which dissolves the wax, leaving the thin slice of tissue attached to the slide, where it can be stained or treated with antibodies or other agents.

After staining, a coverslip is mounted over the tissue using a mounting medium that has the same refractive index as the glass slide and coverslip.

Phase‐Contrast Microscopy

Small, unstained specimens such as a living cell can be very difficult to see with a bright‐field microscope.

The phase‐contrast microscope makes highly transparent objects more visible.

We can distinguish different parts of an object because they affect light differently from one another.

One basis for such differences is refractive index.

Cell organelles are made up of different proportions of various molecules:

DNA,
RNA,
protein,
lipid,
carbohydrate,
salts,
and water.

Regions of different composition are likely to have different refractive indices.

Normally such differences cannot be detected by our eyes.

However, the phase‐contrast microscope converts differences in refractive index into differences in intensity (relative brightness and darkness), which are visible to the eye.

Phase‐contrast microscopes accomplish this result by;

separating the direct light that enters the objective lens from the diffracted light emanating from the specimen
and causing light rays from these two sources to interfere with one another.

The relative brightness or darkness of each part of the image reflects the way in which the light from that part of the specimen interferes with the direct light.

Advantage of phase‐contrast microscopy

Phase‐contrast microscopes are most useful for examining intracellular components of living cells at relatively high resolution.

For example, the dynamic motility of mitochondria, mitotic chromosomes, and vacuoles can be followed and recorded with these optics.

Simply watching the way tiny particles and vacuoles of cells are bumped around in a random manner in a living cell conveys an excitement about life that is unattainable by observing stained, dead cells.

The greatest benefit derived from the invention of the phase‐contrast microscope has not been in the discovery of new structures, but in its everyday use in research and teaching labs for observing cells in a more revealing way.

Limitations of phase‐contrast microscopy

The phase‐contrast microscope has optical handicaps that result in loss of resolution, and the image suffers from interfering halos and shading produced where sharp changes in refractive index occur.

The phase‐contrast microscope is a type of interference microscope.

Other types of interference microscopes minimize these optical artifacts by achieving a complete separation of direct and diffracted beams using complex light paths and prisms.

Another type of interference system, termed Differential Interference Contrast (DIC), or sometimes Nomarski Interference after its developer, delivers an image that has an apparent three‐dimensional quality.

Contrast in DIC microscopy depends on the rate of change of refractive index across a specimen.

As a consequence, the edges of structures, where the refractive index varies markedly over a relatively small distance, are seen with especially good contrast.

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