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Darkfield Illumination : Application, Image Appearance,echnical Details

What is Darkfield Illumination ?

Darkfield Illumination is a transmitted light technique that uses oblique light to illuminate the sample. Light that does not impinge on the sample is not collected by the objective and results in a dark background. Light that interacts with the sample is scattered (refracted, reflected, and/or diffracted) and is “bent” toward the objective collection angle. This light is collected by the objective and is seen as light spots or areas (resulting from scattered light) on a dark background. Contrast is therefore generated and the sample visualized.Darkfield illumination is provided to the sample by a specialized condenser. The simplest DF condenser has a Stop, or Annulus illuminating ring (A). Here, an opaque circle obscures the central portion of the condenser light path. This allows only light in a ring to illuminate the sample. The diameter of the central stop, and thus illuminating annulus, is such that the angle of light is greater than the collecting angle of the objective. Thus without a sample, no light is collected by the objective. This kind of DF stop is useful only for low magnification objectives (<20x).For higher magnification objectives, modifications of the Annular Stop are: B: Immersion paraboloid; C: immersion double mirror concentric; D: cardioid concentric. Gray cone represents the light reflected and refracted from the specimen and collected by the objective. Hatched areas represent glass. Light blocking stops (s) limit light transmission to a hollow cone. i: Immersion oil.; r: reflecting surfaces. (Ruzin,1999).


Optical Microscopy Application: Darkfield Illumination

Darkfield illumination is a technique in optical microscopy that eliminates scattered light from the sample image. This yields an image with a dark background around the specimen, and is essentially the complete opposite of the brightfield illumination technique. The primary imaging goal of the darkfield illumination technique is to enhance the contrast of an unstained sample, which is incredibly powerful, yet simple, for live cellular analysis or samples that have not gone through the staining process.

Darkfield Illumination Image Appearance

A typical darkfield illumination image has a white/bright specimen with a dark background and environment filling the image. This is the exact opposite of a brightfield illumination image, and is useful for unstained specimens or images that require increased contrast. The advantage with using darkfield illumination is that unstained specimens can remain alive and vital, whereas their brightfield counterparts must be treated and are no longer active. Also, it is possible to acquire more qualitative results with this technique through live cellular analysis. For additional information on the brightfield technique, please read Optical Microscopy Application: Brightfield Illumination.

Darkfield Illumination Technical Details

The light path of the darkfield illumination technique is typically applied to an upright microscope, as seen in Figure 2. The light path consists of three key components.
Light Source: enters the microscope and hits the dark field patch stop, which is a disc used to block light from entering the condenser and leaves a circular ring of illumination.Condenser Lens: collects outer ring of illumination and focuses it on the sample.Objective Lens: light hits the sample, and is transmitted or scattered from it. Scattered light enters the objective lens, whereas transmitted light is not collected by the lens. The direct illumination block assists in this step.

What is Darkfield Illumination?

Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the specimen. This light is gathered in the condenser, then shaped into a cone where the apex is focused on the plane of the specimen. In order to view a specimen under a brightfield microscope, the light rays that pass through it must be changed enough in order to interfere with each other (or contrast) and therefore, build an image. At times, a specimen will have a refractive index very similar to the surrounding medium between the microscope stage and the objective lens. When this happens, the image can not be seen. In order to visualize these biological materials well, they must have a contrast caused by the proper refractive indices, or be artificially stained. Since staining can kill specimens, there are times when darkfield microscopy is used instead.

In darkfield microscopy the condenser is designed to form a hollow cone of light , as apposed to brightfield microscopy that illuminates the sample with a full cone of light. In darkfield microscopy, the objective lens sits in the dark hollow of this cone and light travels around the objective lens, but does not enter the cone shaped area. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is placed on the stage it appears bright against a dark background. It is similar to back-lighting an object that may be the same color as the background it sits against – in order to make it stand out.

What is Disadvantages of Darkfield Illumination?

A Darkfield Illumination can result in beautiful and amazing images; this technique also comes with a number of disadvantages.

First, dark field 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 dark field 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 images degradation, flare and distortion and even decrease the contrast and details of the specimen.
Dark field 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 has many applications and is a wonderful observation tool, especially when used in conjunction with other techniques.

However, when employing this technique as part of a research study, you need to take into consideration the limitations and knowledge of possible unwanted artifacts.

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