Dark Field Viewing

This article presents some information about dark field microscopes and describes where and how they are used. According to this article, it is wonder why microscopies using dark field microscopes are not being promoted and used more often. This method is a relatively inexpensive option when it comes to phase contrast optics. The images also appear to be of much better-quality when compared to other contrast and bright field equipment.

When viewing a specimen using dark field microscopes, the article describes briefly how it is done. It also includes illustrations of the dark field microscope and its corresponding parts. An opaque disc is positioned beneath the lens that condenses light. This allows light from the specimen to arrive at the eye. The image that is seen is generally white against a dark backdrop, regardless of what the color of the specimen on the slide may be. False colors can be seen with pigmented objects, meaning that they appear to be of different colors except for their real or actual colors. The overall consensus is that when viewing specimens using dark field microscopy, the resolution or the clarity of the detail of the image is better compared to viewing the same slide under bright field microscopy.

The article states that it is not difficult to achieve a dark field effect using ordinary equipment, although it is advantageous to use the built in occulting disk of the condenser for more dramatic images. Any respectable optical microscope can achieve this type of set-up, though a higher intensity light is necessary to be able to reflect light back to viewer instead of letting light pass directly through the specimen. In a dissecting microscope, the article suggests placing the slide with the specimen to be viewed over the opening so that the light is reflected off the cover slip and the slide itself and not from under the slide. If there is no platform, a stand may be devised to hold the slide. Any built in lights should be turned off. The light source that is high in intensity should be aimed at the specimen at an angle, letting the light pass through a glass pane.

The article also explains in detail how to convert a compound microscope into a dark field microscope. To optimize a good dark field effect, the higher the light intensity is recommended. Students will have a good time trying to “better” their microscopes as they achieve ark field effects through trial and error.

Specimens that look stunning under dark field microscopy include pond water samples. Protozoans, metazoans, algae and other pond organisms are very distinguishable and detailed. With 100x magnification, bacteria is easy seen. Since there are no stains to render the bacteria immobile, live cells and movement can be observed.

Dark field microscopy is recommended for viewing liquid samples. Some clinicians sometimes use this method for live blood cell microscopies. The dark field method is also valuable in viewing cells that are in suspension. Everything within the liquid samples can be effortlessly observed.

ORIGINAL TEXT:

Dark field optics are a low cost alternative to phase contrast optics. The contrast and resolution obtained with inexpensive dark field 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 dark field optics, particularly when cultured with living Paramecium.

Principle

To view a specimen in dark field, 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 (figure 2). 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 dark field as opposed to bright field viewing.

You don’t need sophisticated equipment to get a dark field effect, although the effect is most dramatic when the occulting disk is built into the condenser itself. You do need a higher intensity light, since you are seeing only reflected light. At low magnification (up to 100x) any decent optical instrument can be set up so that light is reflected toward the viewer rather than passing through the object directly toward the viewer.

To set up a dissecting microscope for “dark field” viewing, the specimen should be placed over an opening so that light reflects only from surfaces between cover slip and slide, not from a surface beneath the slide. You may need to make a stand to hold the slide. The surface beneath the opening should be a flat black. Turn off any built-in illuminator. Aim a high-intensity light source toward the specimen at an angle, from the top or side through a glass dish or jar.

With a compound microscope, dark field is obtained by placing an occulting disk in the light path between source and condenser. A cheap set of occulting disks can be prepared by cutting circular pieces of black electrical tape ranging from dime-size up to a diameter that equals the width of the slide, and sticking them to the slide in a row. The circles should be spaced well apart. A specimen is placed on the microscope stage as usual, and the illumination should be made as uniform as possible. If there is an aperture diaphragm in the condenser (contrast lever), it should be opened up wide. After focusing at low power, the slide with occulting disks is placed in the light path between source and condenser, bringing it as close to the bottom of the condenser as it will go.

I would start with the largest disk, sliding it around until it is directly in the center of the light path. Increasing the illumination should then produce a good dark field effect. To optimize, first try stopping down the field diaphragm to get the best contrast between background and specimen. Try to match the size of the occulting disk to the field diameter, so that the edge of the disk is just outside the field of veiw - smaller disks are appropriate for higher power objectives. Vertically, the disk should be a close to the condenser as possible, to make the contrast the greatest. On microscopes with built-in dark field equipment, the view is so impressive because the occulting disk is built into the condenser - very close and focused. After testing the set-up this way, a stand might be rigged to fit under the microscope, so the slide can be placed in position without holding it. Something that ‘grabs’ the condenser and supports the occulting disks would be ideal. The less the students have to mess with, the better.

I set this up on the crummiest little piece of garbage microscope I could find, and it looked very good. A relatively new student-model microscope should give a much better effect.

Suspensions of cells and samples of pond water look spectacular in dark field. While specimens may look washed out and lack detail in bright field, protists, metazoans, cell suspensions, algae, and other microscopic organisms are clearly distinguished and their details show up well. At 100x you can readily see bacteria, even distinguish some structure (rods, curved rods, spirals, or cocci) and movement. Non-motile bacteria look like vibrating bright dots against a dark background. Motile bacteria can be seen moving in a definite direction, sometimes remarkably fast. In pond water samples you may find Spirillum volutans, a very large (up to 0.5 mm) motile spiral bacterium.

When to use dark field illumination

Dark field illumination is most readily set up at low magnifications (up to 100x), although it can be used with any dry objective lens. Any time you wish to view everything in a liquid sample, debris and all, dark field is best. Even tiny dust particles are obvious. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens. Use dark field for

Initial examination of suspensions of cells such as yeast, bacteria, small protists, or cell and tissue fractions including cheek epithelial cells, chloroplasts, mitochondria, even blood cells (small diameter of pigmented cells makes it tricky to find them sometimes despite the color).

Initial survey and observation at low powers of pond water samples, hay or soil infusions, purchased protist or metazoan cultures. Examination of lightly stained prepared slides. ? Initial location of any specimen of very small size for later viewing at higher power. Determination of motility in cultures.

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