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dark field illumination

what is dark field illumination?

what is dark field illumination?

Use scissors or (preferably) a brass cork borer to cut a set of stops matched to all of the objectives, and glue them to a sturdy sheet of clear acetate or glass. The acetate or glass substrate should be easily mountable onto the underside of the substage condenser, either through a filter holder or by other means, such as adhesive tape. Alignment of the stop can be done by observing it through a Bertrand lens or removing the eyepiece and viewing through a phase telescope while adjusting the condenser centering screws.

Darkfield Microscopy at High Magnifications

For more precise work and blacker backgrounds, you may choose a condenser designed especially for darkfield, i.e. to transmit only oblique rays. There are several varieties: “dry” darkfield condensers with air between the top of the condenser and the underside of the slide–and immersion darkfield condensers which require the use of a drop of immersion oil (some are designed to use water instead) establishing contact between the top of the condenser and the underside of the specimen slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great obliquity and free of chromatic aberration, producing the best results and blackest background.

Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of glass ground very accurately into the shape of a paraboloid, as illustrated in Figure 5(b). Light incident upon the reflecting surface (between the glass and condenser housing in Figure 5(b)) of a paraboloid condenser will be focused at the focal point of the reflector. Most paraboloid condensers are cut to ensure that the focal point is slightly beyond the top of the condenser so that parallel light rays will be focused at a position that maximizes illumination of the specimen. The light stop at the bottom of the glass condenser serves to block central rays from reaching the specimen. Light rays that are reflected by the condenser are angled higher than the critical angle of reflection and converge at the principal focus of the condenser. The combination of a glass slide, mounting medium, and immersion oil (between the condenser and the microscope slide) complete the optical homogeneity of the paraboloid shape.

As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures below 0.75 (Figure 5(a)), while the paraboloid and cardioid immersion condensers (Figures 1 and 5(b)) can be used with objectives of very high numerical aperture (up to 1.4). Objectives with a numerical aperture above 1.2 will require some reduction of their working aperture since their maximum numerical aperture may exceed the numerical aperture of the condenser, thus allowing direct light to enter the objective. For this reason, many high numerical aperture objectives designed for use with darkfield as well as brightfield illumination are made with a built-in adjustable iris diaphragm that acts as an aperture stop. This reduction in numerical aperture also limits the resolving power of the objective as well as the intensity of light in the image. Specialized objectives designed exclusively for darkfield work are produced with a maximum numerical aperture close to the lower limit of the numerical aperture of the darkfield condenser. They do not have internal iris diaphragms, however the lens mount diameters are adjusted so at least one internal lens has the optimum diameter to perform as an aperture stop.

Table 2 lists several properties of the most common reflecting high numerical aperture darkfield condensers. This table should be used as a guide when selecting condenser/objective combinations for use with high numerical aperture darkfield applications.
High Numerical Aperture Darkfield Condenser Specifications
Condenser Type Hollow Cone
Numerical Aperture Objective Maximum
Numerical Aperture Number of Reflecting
Surfaces Optical Corrections
Paraboloid 1.00-1.40 0.85 1 Parabolic Achromatic
Cardioid 1.20-1.30 1.05 1 Spherical
1 Cardioidal Achromatic/
Aplanatic
Bicentric 1.20-1.30 1.05 1 Cardioidal
1 Spherical Aplanatic
Bispheric 1.20-1.30 1.05 2 Spherical Aplanatic
Cassegrain 1.40-1.50 1.30 1 Aspheric
1 Spherical Aplanatic
Spot Ring
(Bicentric) 1.40-1.50 1.30 2 Spherical Aplanatic
Nelson
Cassegrain 1.30-1.45 1.20 1 Aspheric
1 Spherical Aplanatic

The condensers illustrated in Figure 5 are designed specifically to produce oblique hollow light cones of high numerical aperture for darkfield illumination. In both instances, the upper surface of the condenser is planar and perpendicular to the optical axis of the microscope. The condenser on the left (Figure 5(a)) is designed to be used “dry” with no oil between the condenser and the underside of the microscope slide. In contrast, the paraboloid condenser in Figure 5(b) is intended to be “oiled” to the bottom of the microscope slide, directly underneath the specimen. Omission of immersion oil when using this condenser (or any of the other condensers listed in Table 2) will preclude any light from reaching the specimen. The oblique hollow cone of light rays emitted by these condensers cannot emerge from the top lens without oil and will be totally reflected back into the condenser. Light emitted from the illumination source is reflected at the mirrored glass surfaces within the interior of the condensers and exits the top of the condensers at much higher angles of inclination than the critical angle (approximately 41 degrees) at which total reflection occurs for passage of light from glass to air. In the situation of the oiled paraboloid condenser (Figure 5(b) and the condensers in Table 2) where the refractive index of the condenser glass, immersion oil, and glass slide are equal, light emitted from the condenser passes through the specimen unrefracted by glass-air interfaces.
Hollow Light Cone Numerical Aperture

Use this tutorial to visualize how the hollow cone of light changes with numerical aperture in reflecting darkfield condensers.
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Reflecting high numerical aperture condensers listed in Table 2 cover a wide range of designs used to produce the oblique hollow cone of light necessary for high-magnification darkfield microscopy. The paraboloid darkfield condenser has been discussed in detail above. Another very useful design is the cardioid condenser that is illustrated in Figure 1. This condenser design utilizes a mirrored hemisphere in the center of the condenser that serves as both a light stop and a reflector to direct light onto a second reflecting surface shaped to resemble a cardioid of revolution, from which the condenser derives its name. The combination of spherical and cardioid reflecting surfaces produces a condenser that is free from coma and both spherical and chromatic aberration. There are several technical drawbacks to using a condenser of such high numerical aperture. The cardioid condenser is very sensitive to alignment and must be carefully positioned to take advantage of the very sharp cone of illumination, making it the most difficult darkfield condenser to use. In addition, the condenser produces a significant amount of glare, even from the most minute dust particles, and the short focal length may result in poor illumination on objects that exceed a few microns in size or thickness. When choosing microscope slides for quantitative high-magnification darkfield microscopy, make certain to select slides made from a glass mixture that is free of fluorescent impurities.

High numerical aperture reflecting condensers (Figures 1, 5, 6 and Table 2) with darkfield illumination provide the method of choice for observing and photographing collections of very small particles or colloidal suspensions, even when the particle diameter is significantly lower than the limit of resolution for the objective. This is due to light diffracted by the particles, which passes through the objective and becomes visible as bright diffraction disks. Each particle is visible as a minute diffraction disk, provided the lateral distance between adjacent particles is greater than the limit of resolving power of the objective. As illumination intensity is increased, the optical difference between minute diffracting particles and their background increases. Simultaneously, even smaller particles (detectable solely by their ability to scatter light) now diffract enough light to become visible and suspended particles can be seen even when their diameters are smaller than 40 nanometers, which is about one-fifth the 200 nanometer resolution limit with oil immersion objectives of the highest numerical aperture. In biological applications, the movements of living bacterial flagella that average about 20 nanometers in diameter (too small to be seen in brightfield or DIC illumination) can be observed and photographed using high numerical aperture darkfield condensers.

Careful attention should be paid to the details of oiling a high numerical aperture condenser to the bottom of the specimen slide. It is very difficult to avoid introduction of tiny air bubbles into the area between the condenser top lens and the bottom of the microscope slide, and this technique should be practiced to perfection. Air bubbles will cause image flare and distortion, leading to a loss of contrast and overall image degradation. Problems are also encountered when using microscope slides that are either too thick or too thin. Many darkfield condensers contain the range of usable slide thickness inscribed directly on the condenser mount. If the slide is too thick, it is often difficult to focus the condenser without resorting to a higher viscosity immersion oil. On the other hand, slides that are too thin have a tendency to break the oil bond between the condenser and the slide. It is a good idea to purchase precision microscope slides of the correct thickness to avoid any of the problems mentioned above.
Darkfield Condenser Adjustment

Explore how alignment and configuration of a darkfield condenser affects image quality.
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A unique situation arises when specimens immersed in aqueous medium are being imaged using a high numerical aperture darkfield condenser. Under these conditions the refractive index of the aqueous solution limits the angle of inclination under which light can pass from the glass microscope slide (n = 1.515) into the water (n = 1.336) surrounding the specimen. The maximum numerical aperture of light passing from glass to water is given by the following equation:

NA (illumination) = 1.555 × sin(i) = 1.336 × sin(90°)

and because sin(90°) = 1

NA (illumination) = 1.336

Even though reflecting darkfield condensers designed for oil immersion are listed with upper limits of numerical aperture as high as 1.50 (see Table 2), light contributing to the illumination of specimens in aqueous media must have a numerical aperture no greater than 1.336, reducing the effective upper limit of darkfield illumination. In the case of specimens immersed in liquids of higher refractive index, the effective upper limit of the numerical aperture of darkfield illumination can approach a maximum of 1.50, although this is difficult to achieve in practice.

High numerical aperture condensers, whether intended for use dry or with oil, must be accurately centered in the optical path of the microscope to realize optimum performance. To achieve this, many darkfield condensers are built with a small circle engraved onto the upper surface to aid in centering the condenser. Centering is performed with a low power (10x-20x) objective by imaging the engraved circle and using the condenser centering screws to ensure the circle (and condenser) are correctly centered in the optical path. For more detailed information about microscope alignment for darkfield illumination, consult our section on darkfield microscope configuration elsewhere in the microscopy primer.

In general, objects imaged under proper conditions of darkfield illumination are quite spectacular to see (e.g. try a drop of fresh blood in darkfield). Often specimens containing very low inherent contrast in brightfield microscopy shine brilliantly in darkfield. Such illumination is best for revealing outlines, edges, boundaries, and refractive index gradients. Unfortunately, darkfield illumination is less useful in revealing internal details.

Other types of specimens, including many that are stained, also respond well to illumination under darkfield conditions. Figure 7 illustrates darkfield photomicrographs of three types of specimen, all of which produce good contrast in both brightfield and darkfield illumination. Details in the body of the deer tick (Ixodes demmini) shown in Figure 7(a) can be washed out in brightfield, unless the condenser aperture is stopped down to maximize contrast. However, in darkfield, most of the specimen detail in the tick becomes visible and can be easily captured on film. The heavily stained helminth trematode (Echinostoma revolutum, Figure 7(b)) also reveals considerably more detail when illuminated under darkfield conditions, as does the silkworm trachea and spiracle illustrated in Figure 7(c). In addition to the examples presented above, a number of other specimens can also be viewed and photographed under both brightfield and darkfield illumination to achieve the desired effects.

During the first half of the Twentieth Century, darkfield microscopy had a very strong following and much effort was expended in optimizing darkfield condensers and illuminators. This intense interest slowly began to fade with the emergence of more advanced contrasting-enhancing techniques such as phase contrast, differential interference contrast, and Hoffman modulation contrast. Recently, a renewed interest in transmitted darkfield microscopy has arisen due to its advantages when used in combination with fluorescence microscopy.

Darkfield microscopy is still an excellent tool for both biological and medical investigations. It can be effectively used at high magnifications to photograph living bacteria, or at low magnifications to view and photograph cells, tissues, and whole mounts. Marine biologists continue to use darkfield illumination at low powers to observe and record data about fresh and salt water organisms such as algae and plankton.

dark field illumination

What is dark field illumination?

What is dark field illumination?

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 technique

When 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

Difference between Dark and Bright Field Illumination

Difference between Dark and Bright Field Illumination

-When you view a particular specimen under a bright field microscope, you will observe that the specimen is dark while its background is bright; hence the name bright field microscope.
On the other hand, when you view a particular specimen under a dark field microscope, you will observe that the specimen is bright while its background is dark; hence the name dark field microscope.
-Since little light actually falls on the specimen, dark-field illumination shows less detail overall than bright-field illumination.

Bright Field Illumination.
A way of illuminating a specimen in a microscope by lighting it from behind, making the specimen appear dark against a bright background. It is considered the most basic type of microscope

The Dark field illumination requires blocking out the central light waves along the optical axis of the light waves. Blocking the light waves allows you to see the specimine when only the oblique rays hit the specimen at an angle.
Parts of the Microscope.
Monitor: To display the picture of the specimen your CMO objective lens is focused on.

CMO Objective lens: To magnify on the part of a specimen you wish to observe.

Stage: To hold your specimen.

Lamp Voltage: Controls how bright the light is.

Zoom Body: To zoom in on the specimen.

Illumination section: Illuminates the specimen.

Brightfield/Darkfield Diascopic Stand. Holds the inner pieces of the microscope inside.

Camera Control: Controls where the CMO Objective lens focuses on.

Economic observation tube: Allows you to see the specimen without the monitor.

Digital camera: Transfers the pixels onto the moniter so you can see the specimen.

Stage: Holds the microscope together and supports the microsope.

dark field illumination

How to Bringing Light to the dark field illumination

Have you ever heard of a dark field microscope? While such a name may sound like a sci-fi gadget used to measure black holes, in reality it’s just a handy tool used to view certain types of translucent samples. The average microscope user may not know about the concept of dark field microscopy, yet it can shed new light on the old way of viewing specimens.

Most people who have survived a biology class know what a light field microscope is. This type of scope uses bright field illumination, meaning it floods the specimen with white light from the condenser without any interference. Thus the specimen shows up as a dark image on a light background (or white field if you will).

This type of unit works best with specimens that have natural color pigments. The samples need to be thick enough to absorb the incoming light; so staining is usually paired with this type of microscope.

Plankton illuminated with a dark field microscopeYet what if the specimen is light colored or translucent, like the plankton on the right? It certainly won’t stand out against a strong white background. Additionally, some specimens are just too thin. They cannot absorb any of the light that passes through them, so they appear invisible to the user. This is where the concept of dark field illumination comes in!

Rather than using direct light from the condenser, one uses an opaque disk to block the light into just a few scattered beams. Now the background is dark, and the sample reflects the light of the beams only. This results in a light colored specimen against a dark background (dark field), perfect for viewing clear or translucent details.

On a grand scale, the same thing happens every day when you look up at the sky. Do the stars disappear when it’s light out? Of course not! They’re still there, their brilliance blotted out by the mid-day sun.

If you’re still having a hard time visualizing this concept, think of a dusty room with the light on and the door open. You may feel the dust affecting your breathing, but you probably won’t see it flying through the air.

Now turn off the light and close the door to just a sliver, while leaving the light on in the adjacent room. If you look at that sliver of light coming through the door, you’ll see all sorts of dust motes suspended in it. You’re employing a similar principle when you use dark field illumination!

dark field illumination

What is Disadvantages of dark field illumination?

What is Disadvantages of dark field illumination?

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.

dark field illumination

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