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sidestream dark field microscopy

sidestream dark field microscopy Conclusion

 

A dark field microscope can offer brilliant, light images against a dark background of otherwise difficult to view specimens.Most standard microscopes come with dark field capabilities or accessories to enable this illumination technique.There are many practical applications of dark field, especially in the field of marine biology, in viewing the many specimens you cannot see using alternative techniques.However, a researcher must keep in mind the potential issues and limitations that may arise from dark field illumination.

sidestream dark field microscopy

How to change Dark Field Transformation?

Most stereo and standard compound microscopes have the potential for sidestream 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.

Too expensive? What you can do…

If you do not have access to these accessories and cannot afford a dark field kit, there are alternative ways to adapt your microscope for dark field illumination.

The expensive stops are all made of opaque material.

Any possible substitutions cannot have any transparent properties.

One option is to use a circular object, such as a coin; adhere the coin to a larger disk and place below the stage.

You can also cut out a round piece of thick paper, such as construction paper, cardboard or poster-board, and attach to the condenser.

Whatever you use, the trick is to find the right diameter so that the makeshift stop will block the light and only allow the oblique rays to illuminate the specimen.

sidestream dark field microscopy

What is sidestream dark field microscopy?

Similar to a bright field but it is modified by a dark field stop just below the source. The dark field stop is just  the condenser, and blocks the light in the center of the lightsource so that the only light that goes through is around the edges. That light is then bent by the condenser and diffacts off the specimen. None of the light goes directly from the light source into the objective, so if there is no specimen, the image will be very dark. The specimen in this method will be illuminated against a black background.
A dark field microscope is useful because it increases the contrast of the image and does not use stains. The lack of staining means that it can be used on live specimens and that one can observe the motility of the organism as well as its correct morphology. Usually, the stains and enzymes used in labs can distort the shape of the organism, but that isn’t an issue with dark field microscopy. This method can also be used to see organisms that are hard to stain, such as Treponema pallidum, spirochetes, and mycoplasma.The one downside is that it’s not possible to see the inclusions, or internal details of the cell

sidestream dark field microscopy

What Advantages of sidestream dark field 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.

sidestream dark field microscopy

sidestream dark field microscopy

 

sidestream dark field microscopy (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.

Light microscopy applications

In optical microscopy, dark-field describes an illumination technique used to enhance the contrast in unstained samples. It works by illuminating the sample with light that will not be collected by the objective lens and thus will not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

The light’s path

The steps are illustrated in the figure where an inverted microscope is used.
Diagram illustrating the light path through a dark-field microscope

Light enters the microscope for illumination of the sample.
A specially sized disc, the patch stop (see figure), blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification.
The condenser lens focuses the light towards the sample.
The light enters the sample. Most is directly transmitted, while some is scattered from the sample.
The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct-illumination block (see figure).
Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.

Advantages and disadvantages

sidestream dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.

The main limitation of dark-field microscopy is the low light levels seen in the final image. This means that the sample must be very strongly illuminated, which can cause damage to the sample. sidestream dark field microscopy techniques are almost entirely free of artifacts, due to the nature of the process. However, the interpretation of dark-field images must be done with great care, as common dark features of bright-field microscopy images may be invisible, and vice versa.

While the dark-field image may first appear to be a negative of the bright-field image, different effects are visible in each. In bright-field microscopy, features are visible where either a shadow is cast on the surface by the incident light or a part of the surface is less reflective, possibly by the presence of pits or scratches. Raised features that are too smooth to cast shadows will not appear in bright-field images, but the light that reflects off the sides of the feature will be visible in the dark-field images.

Use in computing

sidestream dark field microscopy has recently been used in computer mouse pointing devices, in order to allow an optical mouse to work on transparent glass by imaging microscopic flaws and dust on its surface.

sidestream dark field microscopy combined with hyperspectral imaging

When coupled to hyperspectral imaging, dark-field microscopy becomes a powerful tool for the characterization of nanomaterials embedded in cells. In a recent publication, Patskovsky et al. used this technique to study the attachment of gold nanoparticles (AuNPs) targeting CD44+ cancer cells.

Transmission electron microscope applications

Dark-field studies in transmission electron microscopy play a powerful role in the study of crystals and crystal defects, as well as in the imaging of individual atoms.

Conventional dark-field imaging

Briefly, imaging involves tilting the incident illumination until a diffracted, rather than the incident, beam passes through a small objective aperture in the objective lens back focal plane. Dark-field images, under these conditions, allow one to map the diffracted intensity coming from a single collection of diffracting planes as a function of projected position on the specimen and as a function of specimen tilt.In single-crystal specimens, single-reflection dark-field images of a specimen tilted just off the Bragg condition allow one to “light up” only those lattice defects, like dislocations or precipitates, that bend a single set of lattice planes in their neighborhood. Analysis of intensities in such images may then be used to estimate the amount of that bending. In polycrystalline specimens, on the other hand, dark-field images serve to light up only that subset of crystals that are Bragg-reflecting at a given orientation.

Weak-beam imaging

Weak-beam imaging involves optics similar to conventional dark-field, but use of a diffracted beam harmonic rather than the diffracted beam itself. Much higher resolution of strained regions around defects can be obtained in this way.

Low- and high-angle annular dark-field imaging

Annular dark-field imaging requires one to form images with electrons diffracted into an annular aperture centered on, but not including, the unscattered beam. For large scattering angles in a scanning transmission electron microscope, this is sometimes called Z-contrast imaging because of the enhanced scattering from high-atomic-number atoms.

Digital dark-field analysis

This a mathematical technique intermediate between direct and reciprocal (Fourier-transform) space for exploring images with well-defined periodicities, like electron microscope lattice-fringe images. As with analog dark-field imaging in a transmission electron microscope, it allows one to “light up” those objects in the field of view where periodicities of interest reside. Unlike analog dark-field imaging it may also allow one to map the Fourier-phase of periodicities, and hence phase gradients, which provide quantitative information on vector lattice strain.

sidestream dark field microscopy

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