What is Darkfield Microscopy?

What is Darkfield Microscopy?

Darkfield microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

This microscopy technique takes advantage of oblique illumination to enhance contrast in specimens that are not imaged well under normal illumination conditions.

In optical microscopes, a darkfield condenser lens must be used, which directs a cone of light away from the objective lens.

After the direct light has been blocked by an opaque stop in the condenser, light passing through the specimen from oblique angles is diffracted, refracted, and reflected into the microscope objective to form a bright image of the specimen superimposed on a dark background.

This dark background provides a high degree of contrast and can make samples with difficult backgrounds stand out with relatively little effort.

To maximize the scattered light-gathering power of the objective lens, oil immersion is used and the numerical aperture (NA) of the objective lens must be less than 1.0.

Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the numerical aperture (NA). Often these objective lenses have a numerical aperture (NA) that is variable from 0.7 to 1.25.

Darkfield optics are a low-cost alternative to phase-contrast optics. The contrast and resolution obtained with inexpensive darkfield equipment may be superior to what you have with student-grade phase contrast equipment. It is surprising that few manufacturers and vendors promote the use of dark field optics.

A dilute suspension of yeast cells makes a good practice specimen for darkfield optics, particularly when cultured with living Paramecium.

Working Principle Darkfield Microscopy

The dark field of microscopy is the principle of making transparent microscopic objects visible. To view a specimen in a darkfield, an opaque disc is placed underneath the condenser lens so that only light that is scattered by objects on the slide can reach the eye. Instead of coming up through the specimen, the light is reflected by particles on the slide.

Everything is visible regardless of color, usually bright white against a dark background. Pigmented objects are often seen in “false colors,” that is, the reflected light is of a color different than the color of the object. Better resolution can be obtained using darkfield as opposed to bright field viewing.

The condenser lenses of the microscope focus the light toward the sample being examined. By using a darkfield condenser, the light is scattered in all directions.

The dispersed light is then blocked out by the condenser, allowing only the scattered beams of light to reflect back to the eye. This makes the examined specimen appear on a dark background.

Using this method requires you to have darkfield condensers. These are the condensers that refract the non-direct light.

Therefore, it is important to make sure the numerical aperture of the darkfield condenser is larger than the objective lenses you use. Failure to do so will allow direct light to enter your objective lenses, which disrupts the darkfield effect.

There are two types of darkfield condensers: dry and immersion. Dry can focus on specimens that require less magnification, producing a dark field image on lenses with a numerical aperture of .65 or less. For specimens that require greater magnification, an oil immersion condenser is needed to produce a high-quality dark background image.

How Does Darkfield Microscopy Work In TEM?

Aside from the conventional light microscope, the principles of dark field microscopy can also be applied to electron microscopy more specifically, transmission electron microscopy. This is highly important in imaging and studying crystals and atoms.

This works by mapping the intensity of the electron beams diffracted from the specimen in relation to the projected position of this specimen. As a result, when it comes to crystals, their reflective capability becomes pronounced, or lattice defects and bending are identified.

Actually, this conventional dark field technique in electron microscopy is just one of many. Other techniques include weak beam imaging, digital dark field analysis, and low or high-angle annular darkfield imaging.

Critical Angle In Darkfield Microscopy

The critical angle is important in darkfield. The darkfield condenser produces a very oblique angle of light. If this angle is greater than the critical angle at any interface, the illumination will be totally internally reflected. For this reason, the specimen’s immersion medium is important.

The critical angle for glass-to-air is 41 degrees and for glass-to-water, it is 61 degrees. Low-power darkfield condensers work fine for specimens in water.

A high-power darkfield condenser may not be useful with a water-immersed specimen for the above reason. It is always best to immerse the darkfield condenser in the slide with oil even for low-power darkfield condensers as long as the critical angle is not exceeded.

When to Choose Darkfield Microscopy

  • Darkfield Microscopy imaging is used to look at live cells and small organisms such as water fleas or larvae.
  • Darkfield Microscopy is less damaging to live samples as the lower level of light means the sample does not get subjected to as much heat as in BF.
  • Darkfield Microscopy samples are unstained and relatively transparent.
  • Darkfield Microscopy is used for looking at the overall external structure of the sample. Some internal features will be visible depending on the size and opacity of the sample.

Uses of Darkfield Microscopy

This method is great for viewing things that are unstained, transparent, and absorb very little light. Therefore, this method illuminates objects that would otherwise be very hard to see. It is also best for examining specimens at low magnification. This is a great method to use for liquid samples, even those with debris in them.

Here are a few other examples of what dark field microscopy is used for:

Darkfield microscopy is used to study marine organisms such as algae, plankton, diatoms, insects, fibers, hairs, yeast, and protozoa as well as some minerals and crystals, thin polymers, and some ceramics. Darkfield is used to study mounted cells and tissues.

Examining thin bacteria. Bacteria under normal conditions of microscopy will not all appear in the objective lenses since some appear larger. With the darkfield condenser, they will be more visible.

Even microscopic dust particles can be examined on a dark background, producing stunning illuminated images.

Basically, the tinier the sample, the better it is to use dark field illumination. This is similar to stars. Stars are billions of light-years away and appear to be relatively small.

Yet on the dark background of the night sky, we can see them. The same is true in darkfield microscopy; small objects are best viewed on a dark background.

Advantages of Darkfield Microscopy

Darkfield microscopy has many advantages. Its dark background offers a high degree of contrast, making it easy to see samples on difficult backgrounds. This technique is easily accessible since many brightfield lab microscopes can be configured for darkfield illumination.

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 from other illumination techniques.

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

You can also use darkfield microscopy in the research of live bacteria, 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.

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

The disadvantage of Darkfield Microscopy

A dark field microscope can result in beautiful and amazing images; this technique also comes with a number of disadvantages.

  • First, darkfield images are prone to degradation, distortion, and inaccuracies.
  • A specimen that is not thin enough or its density differs across the slide, may appear to have artifacts throughout the image.
  • The preparation and quality of the slides can grossly affect the contrast and accuracy of a darkfield image.
  • You need to take special care that the slide, stage, nose, and light source are free from small particles such as dust, as these will appear as part of the image.
  • Similarly, if you need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles.
  • These liquid bubbles will cause image degradation, flare, and distortion and even decrease the contrast and details of the specimen.
  • Darkfield needs an intense amount of light to work. This, coupled with the fact that it relies exclusively on scattered light rays, can cause glare and distortion.
  • It is not a reliable tool to obtain accurate measurements of specimens.
  • Finally, numerous problems can arise when adapting and using a dark field microscope. The amount and intensity of light, the position, size, and placement of the condenser and stop need to be correct to avoid any aberrations.

Dark field microscopy principle