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The Best Mistake I Ever Made in dark field illumination

dark field illumination

How to Make a dark field illumination

You don’t need to buy a huge expensive set-up to experiment with dark field illumination.

To create a dark field, an opaque circle called a patchstop is placed in the condenser of the microscope. The patchstop prevents direct light from reaching the objective lens, and the only light that does reach the lens is reflected or refracted by the specimen. Easy enough, right?

If you want to make a dark field microscope you’ll first need a regular light microscope. Below is your full list of “ingredients”:

Dark field microscopeMicroscope
Hole punch
Black construction paper
Transparency film
Now use the following steps to make your patchstop:

Set up your microscope and choose the lowest-power objective lens.
Set the eyepiece aside somewhere safe.
Open the diaphragm as wide as possible. Then slowly close it until is just encroaches on the circle of visible light.
Now bend over and take a look at the diaphragm from below. See that opening? It’s only slightly smaller than the finished patchstop you’ll create.
Punch a few circles in the black construction paper with the hole punch. Measure one against the diaphragm opening. If it’s more than 10% larger, cut it down to about that size (10% larger than the diaphragm opening). If it’s smaller, cut out a larger circle.
Cut a 5 cm square of transparency paper.
Glue the black circle onto the transparency film, about 2 cm from the corner of the square. In that free 2 cm of paper, write the correct magnification power of your objective.
Mark the patchstop with the correct magnification power.
Repeat the above steps for all the objective powers except the oil immersion lenses.
Now use your patchstop to turn a light field unit into a dark field microscope:

Select the correct patchstop for the objective power to be used.
Slip the patchstop between the filter holder and condenser. If your microscope has no filter, hold it manually below the condenser.
Remove the eyepiece.
Open the diaphragm and move the patchstop until the light is blocked entirely. Use tape to secure it if there is no condenser on your microscope.
Replace the eyepiece and examine the sample.
As you can see, a dark field microscope can let users see specimens in a whole new way, bringing those into focus that don’t stand out under intense light. Using dark field illumination can open up a whole new view of microscopy
The first picture of the plankton was taken by Uwe Kils and is from Wikipedia under the GNU Free Documentation License.

dark field illumination

When to Use a Dark Field Microscope?

dark field illumination are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens

Single-celled organisms

Live blood samples

Aquatic environment samples (from seawater to pond water)

Living bacteria

Hay or soil samples

Pollen samples

Certain molecules such as caffeine crystals (right)

dark field illumination makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

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

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

How to made the dark field illumination?

How to made the dark field illumination ?

It is very easy to make dark field illumination yourself. What you have to do is place an opaque round stop in the condenser. An easy way is to cut a piece of black paper and put it on a filter in your filterholder. You can put the stop on a piece of clear acetate sheet. You can even try to draw the stop on it with black paint. The most important thing is to have it big enough to stop all light going directly into the objective. Only the light that is reflected by the objects in the sample reaches the objective then. Stronger objectives are more difficult because their NA is often too high. The NA of your condenser should always be higher then the NA of the objective. If patch-stops of 8, 10, 12 and 15mm are made you can’t go wrong really. For objectives of around x10 the middle sizes prove best.If you like to make the patchstop as precise as possible: The best way is to set up as normal (brightfield), remove the eyepiece and close/open the substage iris until it is *just* visible. Then, either bending your neck over double, or carefully removing the condenser, measure the diameter of the iris diaphragm as it is now set. A pair of calipers is useful here. This diameter is that for the patch stop. Very often, to be on the safe side it is best to add about 10% to this figure, this avoids leakage, especially if you have no means of centering the stop in the filter holder. If you have a phase contrast condenser, the largest phase contrast annuli often make excellent patch stops for darkfield!The real connoisseurs must have recognized the skills of Klaus Kemp in the arranged (cleaned) diatom slide photographed by Mike Samworth.

dark field illumination

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