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Why I’m no longer satisfied with bright field vs dark field — and what I’m doing about it.

bright field vs dark field

What is bright field vs dark field?

dark field microscopy of sugar crystals
Dark Field illumination is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

Pictured right: Highly magnified image of sugar crystals using darkfield microscopy technique

When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

bright field vs dark field

What is Advantages of bright field vs dark field?

 

A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light.

These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques.

You can use dark field to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics.

You can also use dark field in the research of live bacterium, as well as mounted cells and tissues.

It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

Dark field microscopy is often dismissed for more modern observation techniques such as phase contrast and DIC, which provide more accurate, higher contrasted images and can be used to observe a greater number of specimens.

Recently, dark field has regained some of its popularity when combined with other illumination techniques, such as fluorescence, which widens its possible employment in certain fields.

bright field vs dark field

What does my scope need to do bright field vs dark field?

What does my scope need to do bright field vs dark field?

Most stereo and compound microscopes can do bright field vs dark field imaging. Check your microscope’s specifications to see if this is your case. If your microscope does not have a built-in condenser or stop, don’t worry, you can probably still use your microscope for bright field vs dark field imaging. You should be able to purchase an aftermarket condenser or even make your own stop. Read below to learn more about condensers and stops.

Condensers-In a bright field vs dark field set-up, an Abbe bright field vs dark field condenser is mounted below the microscope stage. This controls the light before it enters your specimen and objective. It’s made up of two uncorrected lenses and an iris diaphragm. The top lens of an Abbe bright field vs dark field condenser is concave, therefore the light emerging from this top lens forms an inverted hollow cone of light. Subsequently, only oblique light rays reach your specimen. If the numerical aperture of your condenser is greater than your objective these oblique light rays will cross and miss your objective making the background appear dark while reflecting and refracting off your specimen. You can adjust your condenser for optimal brightness, contrast, depth of field, etc two different ways:1) By moving it closer or further away from the specimen and stage, or 2) By opening or closing its iris or diaphragm.

Stops-Stops are opaque discs located just under the bottom lens of the substage condenser. When using stops, both the aperture and field diaphragms need to be opened wide to allow oblique rays to diffuse around the stop and reach your specimen. (Think solar eclipse, where the stop is the moon blocking the earth/specimen from direct light.)  You can purchase stops for almost any scope, or even make your own, by mounting a coin (or other opaque disc) on a clear glass disk.

When is bright field vs dark field good to use?

Dark field is useful when you would like to view unstained, transparent specimens. The best specimens for bright field vs dark field should have a refractive index that is close to the surroundings and otherwise difficult to image using conventional bright field microscopy. For example, many small aquatic organisms have refractive indices that are very similar to their surrounding water, making them ideal candidates for bright field vs dark field microscopy. Other ideal biological candidates include diatoms, small insects, unstained live bacteria, yeast, tissue culture cells, etc. Non-biological candidates include mineral and chemical crystals, and thin sections of polymers.

bright field vs dark field

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