dark field illumination microscopy

What is dark field illumination microscopy?

 

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 (see illustration below), 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.Darkfield microscopy is a simple and popular method for rendering unstained and transparent specimens clearly visible. Good candidates for darkfield observation often have refractive indices very close in value to that of their surroundings and are difficult to image with conventional brightfield techniques. As an example, small aquatic organisms, oocytes, and cells in tissue culture have a refractive index ranging from 1.2 to 1.4, resulting in a negligible optical difference from the surrounding aqueous medium (refractive index of 1.3). These and similar specimens are ideal candidates for observation with darkfield illumination techniques.

The configuration presented in Figure 1 illustrates a modern Nikon SMZ1500 stereomicroscope equipped with an advanced stand containing provisions for both brightfield and darkfield illumination through a clear glass stage mounted on the top of the stand. Also depicted is a digital Internet camera system (Nikon Dn100) capable of transferring images collected by the microscope to remote observers. Details of the darkfield illumination mechanism are discussed below.Illumination of specimens by darkfield requires blocking out of the central light rays along the optical axis of the microscope, which ordinarily pass through and around (surrounding) the specimen. Blocking these light rays allows only those oblique rays originating at large angles to strike the specimen positioned on the microscope stage. In a compound microscope equipped with a simple condenser system, the condenser (Abbe-style) top lens is spherically concave, enabling light rays emerging from the surface in all azimuths to form an inverted hollow cone of illumination having an apex centered in the specimen plane. If no specimen is present on the stage, and the numerical aperture of the condenser is greater than that of the objective, the oblique rays cross and miss entering the objective front lens because of their obliquity.

The field of view will appear dark.Figure 2 – Nikon Diascopic Darkfield and Brightfield Illumination Stand The stereomicroscope illustrated in Figure 1 produces an oblique cone of illumination using a specially-designed seven-sided toroidal mirror (Figure 2) that substantially reduces the stray light entering the large common main objective front lens. The toroidal mirror operates in a manner similar to high numerical aperture reflecting darkfield condensers that are equipped with internal mirror surfaces having a variety of curvature geometries.

What is dark field illumination microscopy?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 techniqueWhen 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.

dark field illumination microscopy Transformation-Most stereo and standard compound microscopes have the potential for dark field microscopy.If a microscope has built-in elements to easily modify for dark field illumination, the manufacturer usually lists this amongst the observation specifications.You can achieve dark field by using condensers, mirrors and/or a “stop.” Some microscopes come with these accessories or researchers can purchase dark field kits, or even use some common items to adapt a microscope for dark field illumination.In bright field illumination, the object is lit from below the stage, resulting in a larger, contrasted image that can be studied.A dark field microscope blocks this central light with a condenser so that only oblique rays hit the object.An Abbe condenser, for example, contains a concave orb that collects light rays in all azimuths that bounce off a sample to form a cone of illumination.If there is nothing on the stage, the aperture of the condenser is greater than the objective and the view will be completely black.A stop is an opaque object that blocks the central light when placed underneath the stage condenser.This also causes light to scatter in all azimuths, resulting in a cone of light that allows for dark field observation.

Advantages of dark field illumination microscopy-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.

Disadvantages of dark field illumination microscopy-A dark field microscope 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.

 

CATEGORY AND TAGS:

dark field microscope, dark field microscopy, darkfield microscope, darkfield microscopy

, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

Related Items

  • Product Categories

  • Recent Posts

  • Archives

  • Categories

  • dark field microscope uses visible light dark field microscopy dark field microscopy analysis dark field microscopy blood dark field microscopy blood cells dark field microscopy blood images dark field microscopy condenser dark field microscopy disadvantages dark field microscopy for live blood analysis dark field microscopy how it works dark field microscopy images dark field microscopy live blood analysis dark field microscopy magnification dark field microscopy pdf dark field tem microscope
  • Translate: