Darkfield Microscope: Definition, Principle, Application.

The concept of darkfield microscopy dates back to the late 19th century, when German scientist Adolf Fick first described the technique in his book “Die Mikroskopie des Lebenden Auges” (The Microscopy of the Living Eye). Fick used a simple microscope with a special condenser lens to illuminate samples from the side, creating a dark background behind the sample.

Over the next few decades, darkfield microscopy was further developed and refined, and the technique became more widely used in the study of biological samples. In the 1930s, American scientist Calvin S. Snyder developed a special darkfield condenser for use in microscopy, which made it easier for researchers to use the technique in their studies.

Today, darkfield microscopy is widely used in a variety of fields, including biology, medicine, and materials science. It is a valuable tool for studying samples that are transparent, have low contrast, or are difficult to observe with other types of microscopy.

What is a Darkfield Microscope?

• A darkfield microscope is a type of microscope that uses darkfield illumination to observe samples. In darkfield microscopy, the sample is illuminated from the side or back, rather than from above, as in a traditional brightfield microscope. This creates a dark background behind the sample, making it easier to see details and structures that might be difficult to see with a brightfield microscope.
• Darkfield microscopy is particularly useful for observing samples that are transparent or have low contrast with the background, such as living cells or small organisms. It can also be used to study samples with a high refractive index, such as crystals or minerals.
• To use a darkfield microscope, a special condenser lens is used to focus the light on the sample from the side or back. This creates a halo of light around the sample, which is then focused onto the eyepieces or a camera by the objective lens. The resulting image shows the sample as a bright object against a dark background, making it easier to see details and structures that might be otherwise difficult to see.
• Darkfield microscopy is commonly used in biological and medical research, as well as in the study of materials science and other fields. It is a useful tool for studying samples that are difficult to observe with other types of microscopy, such as brightfield or fluorescence microscopy.
• Darkfield microscope allows a viewer to observe the specimen bright whereas the surrounding appears as dark.
• Using a dark field microscope we can see the living and unstained cells.
• The darkfield microscope can reveal considerable internal structure in microorganism.
• For a dark field microscope, we can use three types of condenser such as abbe condenser, cardioids condense, paraboloids condenser.
• The condenser lens of a dark field microscope creates a hollow cone of light which is opposite of a bright field microscope.
• An opaque disc is located underneath the condenser lens, which only allows the scattered lights from the object, and prevents the straight lights which are transmitted through the specimen. So that we can see a dark background and the bright object.
• Most of the compound and stereo microscope can be converted into dark field microscope.

Principle of Darkfield Microscope

Dark-field microscopy employs a light microscope with an extra opaque disc beneath the condenser lens or a modified condenser with a central blacked-out section, which prevents light from the source from entering the objective directly.

The light is intended to pass through the outer edge of the condenser at a wide angle and strike the sample at an angle. For visualisation, only the light scattered by the sample reaches the objective lens. All extraneous light that goes through the specimen and misses the target illuminates the specimen brightly against a black background.

Types of Specimens for Dark Field

The finest specimens for dark field are those with scattered refractive objects separated by empty space. If there are too many items or if a thick solid specimen emits light into the microscope, there will be no dark field. Very thin histological sections can be utilised if they are unstained or very partially stained, such as with silver stains. Dark field microscopy is applicable to fluids from animals and plants, cell cultures, microorganisms, edibles, fibres, crystals, colloids, and submicroscopic particles. Also acceptable are autoradiography and gold labelling preparations.

What is Critical Angle In Dark Field Microscopy?

In a dark field, critical angle is vital. The dark field condenser generates an extremely oblique light angle. If this angle exceeds the critical angle at any interface, the light will be completely internally reflected. The specimen’s immersion medium is crucial for this reason. The critical angle for glass to air is 41 degrees, whereas it is 61 degrees for glass to water. Low-power dark field condensers work well for water-based specimens. Due to the aforementioned reason, a high-powered dark field condenser may not be useful with a water-immersed specimen. Even with low power dark field condensers, it is always preferable to immerse the condenser in oil as long as the critical angle is not exceeded.

Parts of Darkfield Microscope

1. Dark Field Condensers

Dark field is the only non-Kohler lighting broad field method we shall investigate. In dark field microscopy, the objective lens receives no direct light from the condenser. The objective admits only light that is reflected, refracted, or diffracted by the specimen. The dark field condenser emits a bright ring. The angle of the light relative to the surface of the slide is highly oblique.

This oblique light is concentrated on the specimen. It then diverges so dramatically that no direct light can penetrate the objective. This sort of illumination is a cone of light that is hollow. The old microscopists employed only one source of oblique illumination to resolve N. Spencrii. Strongly tilting the mirror of the microscope to one side generated this effect.

The dark field condenser illuminates the specimen obliquely from all 360 degrees. To prevent direct light from entering the objective lens, the numerical aperture of the condenser must be greater than that of the objective lens. This is not an issue for dry objectives with low magnification when a 0.95 NA condenser is utilised. However, this is a concern for NA goals with a high priority. Here, the condenser must have a very high NA, such as 1.45, and must be utilised with a maximum NA objective of 1.25.

a. Low Magnification Dark Field Condensers

A low magnification dark field condenser is only an ordinary brilliant field condenser with an opaque disc of the appropriate diameter put in its front focal plane. The opaque disk’s diameter must be precisely sufficient to prevent any direct light from entering the objective. Obviously, this means that objectives with a higher NA require larger drives.

Numerous microscope manufacturers provide “universal” condensers with one or more dark field discs that are compatible with objectives of varying numerical aperture (NA). By increasing or lowering the condenser, one can increase or decrease the apparent size of an opaque disc. Thus, a single disc may be useful for a limited number of objective NAs. If the condenser has a numerical aperture (NA) more than 0.95, better results can be obtained at low magnification if the condenser is lubricated to the slide, even if the objective is used dry. As follows, you can create your own dark field opaque disc for low power objectives:

1. Set up Kohler illumination.
2. Observe the back focal plane of the objective.
3. Adjust the aperture iris till it just fits outside of the objective aperture.
4. Measure the diameter of the aperture iris.
5. Create a disc of this diameter that is opaque.
6. Place the disc as close to the condenser iris as feasible beneath the condenser.
7. If the field is grey, the disc is too tiny, and if the specimen cannot be lighted, the disc is too large.

b. High Numerical Aperture Dark Field Condensers

In 1855, Francis H. Wenham and George Shadbolt developed the first condenser designed exclusively for dark field. Using a parabolic glass reflector, this condenser produced a hollow cone of light. A reflecting condenser does not cause chromatic aberrations, unlike a refracting condenser, and its parabolic shape lowers spherical aberrations. The end result is a more tightly concentrated point of light. Over time, further dark field condenser designs arose. Figure 9.3 and Table 9.1 illustrate some of the most prevalent.

The requirement to keep the objective NA below the condenser NA led to the manufacturing of ever-higher NA condensers and the development of the “funnel stop” for objective lenses. A funnel-shaped cone is put into the objective lens to restrict its aperture.

Modern objective lenses do not utilise funnel stops. Instead, certain high NA oil goals incorporate an iris diaphragm. This iris decreases the NA of the objective lens. Always lubricate high magnification dark field condensers with the specimen slide. Due to the fact that the angle of incidence of light exiting the top of the condenser is far greater than the critical angle for glass to air, no light will emerge from the condenser unless immersion oil is applied to its surface.

2. Eyepiece

The eyepiece is the small lens through which researchers and scientists can see images of utilised biological specimens.

3. A Light Source

Important for the magnification of biological specimens is a light source. If there is a light source, the dark field microscope’s condenser scatters the light rays. Light scattering is entirely dependent on the light source. The only light source in electron microscopy techniques is high-accelerating electrons. The electrons contribute to the process of light ray scattering.

4. Objective Lens

The objective lens of a dark field microscope is located in the dark hollows of the apex cone.

Path of light in Darkfield Microscope

1. Light enters the microscope to illuminate the specimen.
2. A special disc, called patch stop blocks some light from the light source, leaving an outer ring of illumination.
3. A wide phase annulus can also be reasonably substituted at low magnification.
4. Now the condenser lens focuses the light towards the specimen.
5. The light enters the specimen.
6. Most of the light rays are directly transmitted, while some of them are scattered from the sample.
7. Only Scattered light started to enter the objective lens and creates an image of the specimen.
8. While the directly transmitted light simply misses the lens and is not collected due to a direct-illumination block and they omitted.

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Methods for Dark Field Microscopy

In this section, certain dark field techniques and pitfalls are discussed.

Prerequisites for dark field

A few simple conditions eliminate the majority of the difficulties encountered in dark field microscopy:

1. Ensure that the numerical aperture of your condenser is greater than that of the highest-powered objective you will employ.
2. A dark field necessitates an extremely bright light source. Remove all colour and neutral density filters from the light source.
3. Ensure that your glass slides are neither too thick nor too thin, particularly if you are using a dark field condenser with a high NA. High NA dark field condensers function optimally at a fixed focal length. The manufacturer may suggest a specific thickness for the slides, or this value may be printed on the condenser. Standard slides have a thickness of 1.2 mm, but slides from the same box can vary in thickness. The thickness of the slide should be within 0.05 mm of the acceptable value. The correct slide thickness for your condenser can be determined as follows:
   1. Place a slide, with one end frosted on one side, in oil contact with the condenser, with the frosted side towards the goal, while the condenser is in its highest position.
   2. Examine the frosted surface with a 10x objective of low magnification.
   3. The slide is either too thick or too thin if the image displays a brilliant spot with a black centre. If there is only a small bright spot, the thickness of the slide is correct.
   4. If the slide is incorrect, attempt to lower the condenser. If the black centre vanishes and only a small brilliant area remains, the slide is too thin; otherwise, it is too thick.
   5. Check the thickness of the slide with a micrometre and repeat this procedure with slides of varying thicknesses until you reach the optimal thickness.

Centering the dark field condenser

If you are utilising a universal condenser, centering it for Kohler illumination in brilliant field ought to suffice for its dark field configuration. If you are utilising a dark field condenser with a high NA, perform the steps below:

1. Choose an objective with a very low magnification (e.g., 2X or 5X).
2. Provide as much light as feasible.
3. Lubricate the condenser and raise it to the top position beneath a blank slide.
4. If the condenser has a small, brilliant central ring, focus the microscope on it and centre it using the condenser’s centering adjustments. The item in question is a spot ring condenser.
5. If there is no ring, proceed as follows:
   1. Use a slide with the appropriate thickness and one end frosted.
   2. Coat the non-frozen side of the condenser with oil.
   3. Consider the frosty side.
   4. With the condenser’s centering screws, position the bright spot of light in the centre of the condenser. It may be essential to make a minor adjustment to the centering screws when switching to high NA targets.
   5. Assuming that all of your slides have the same thickness, you should not adjust the condenser’s vertical position.

Focusing in dark field at high NA

Since the field is black at high magnification and high NA, it may be difficult to first focus on the specimen. This is particularly true if the sample is sparse. In the case of a highly dispersed specimen, a wax pencil mark on the specimen side of the slide might be used as an initial focusing reference point. The following approach will aid concentration:

1. Lubricate the condenser slide.
2. Lower the stage (or elevate the aim) and lubricate the slide’s top.
3. Raise or lower the stage until the objective and oil are just touching.
4. Examine the microscope; you should observe a bright, diffuse field.
5. First, increase the distance between the microscope slide and the objective lens (if you are already too close), and then decrease it. As you approach focus, the field will first become extremely bright, then grey, and finally black.
6. At the point of sharpest focus, the field will be black and the specimen will sparkle.

Application of Darkfield Microscope

1. The darkfield microscope is used to identify bacteria like thin and distinctively shaped.
2. We Can observe the living and unstained cells by using a dark field microscope.
3. Considerable internal structure in microorganisms can be revealed by the dark field microscope.
4. A biological darkfield microscope used to observe the blood cells.
5. Used to identify algae.
6. A metallurgical darkfield microscope is used to observe hairline metal fractures.
7. Stereo darkfield microscope or gemological microscope used to study the diamonds and other precious stones.
8. A stereo dark-field microscope used to observe the shrimp or other invertebrates.
9. In the study of crystals and imaging of individual atoms, Dark-field studies in transmission electron microscopy play a powerful role.
10. It helps to characterize the embedded nanomaterials in cells by combined with hyperspectral imaging.

Advantages of Darkfield Microscope

1. No need to stain the specimen.
2. It is useful for those specimens that are transparent and absorb little or no light.
3. Marine organisms such as algae, plankton, diatoms, insects, fibers, hairs, yeast, and protozoa can observe under a dark-field microscope.
4. Dark-field microscope can use to study for minerals and crystals, thin polymers, and some ceramics.
5. It is useful to study the external structure of the specimen in great detail.
6. It can be used for the research of live bacteria and mounted cells and tissues.

Disadvantages of Dark-field Microscope

1. Dark-field microscope creates prone to degradation, distortion, and inaccuracies images.
2. If the specimen’s density differs across the slide or is not thin enough, it can create artifacts throughout the image.
3. The slides of bad quality can grossly affect the contrast and accuracy of a dark field image.
4. Before use make sure slide, stage, nose, and light source are free from dust.
5. An intense amount of light is required for a dark field microscope to work.
6. It cannot measure the specimen accurately.
7. Liquid bubbles can be formed during uses of oil or water on the condenser and/or slide, which is almost impossible to avoid.

Challenges of Using Darkfield Microscopy

When preparing specimens for darkfield microscopy, care must be given since characteristics above and below the plane of focus can scatter light and degrade the image. When imaging, the cleanliness of the slide is a critical factor, but in darkfield, it is even more crucial because every piece of debris will be highlighted and can obscure the image.

Other obstacles you may encounter when configuring a microscope for darkfield include:

• Insufficient illumination to render the specimen visible, or the specimen is visible but extremely dim. Darkfield microscopy requires a great deal of light since so much of it is obscured by the cone. Don’t be hesitant to increase the power, but keep in mind that some samples may not tolerate lengthy exposure to this quantity of light.
• There is a dark area in the centre of the field of view, but peripheral objects are well-lit and appear normal. This is typically caused by incorrect condenser alignment or focus. This issue will be resolved by aligning and focussing the condenser. This solution applies to the majority of situations where the sample is in focus but the illumination appears uneven.
• The backdrop has colours, or the background is unevenly lighted with a grey cast. This occurs frequently when the sample is excessively thick or mounted in contaminated media. Remounting the sample ought to be useful.

Why Is Darkfield Microscopy a Good Imaging Technique?

Darkfield microscopy is a good imaging technique because it allows researchers to observe details and structures in a sample that might be difficult to see with other types of microscopy, such as brightfield or fluorescence microscopy.

Some specific advantages of darkfield microscopy include:

1. High contrast: By illuminating the sample from the side or back, darkfield microscopy creates a bright image of the sample against a dark background, making it easier to see details and structures that might be difficult to see with other techniques.
2. Improved resolution: The high-contrast image produced by darkfield microscopy can help to improve the resolution of the image, allowing researchers to see smaller details and structures in the sample.
3. Good for transparent samples: Darkfield microscopy is particularly useful for studying transparent samples, such as living cells or small organisms, which can be difficult to see with other techniques.
4. Can be used with a variety of samples: Darkfield microscopy can be used with a wide range of samples, including biological, mineral, and materials science samples.

Overall, darkfield microscopy is a valuable tool for researchers looking to study samples that are transparent, have low contrast, or are difficult to observe with other techniques. It can provide high-contrast, high-resolution images that can help researchers to better understand the structure and properties of their samples.

Darkfield Microscope Images

Reference

• https://www.microscopemaster.com/dark-field-microscope.html
• https://en.wikipedia.org/wiki/Dark-field_microscopy
• https://www.microscopyu.com/techniques/stereomicroscopy/darkfield-illumination
• https://www.slideshare.net/abhishekindurkar/dark-field-microscopy-81857005
• https://bio.libretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless)/3%3A_Microscopy
• https://www.ruf.rice.edu/~bioslabs/methods/microscopy/dfield.html