dark and bright field microscopy

What is bright field microscopy?

Bright field microscopy is the conventional technique. It is suitable for observing the natural colors of a specimen or the observation of stained samples. The specimen appears darker on a bright background.

What is dark field microscopy?

Darkfield microscopy shows the specimens bright on a dark background. Brightfield microscopes that have a condenser with a filter holder can be easily converted to darkfield by placing a patch stop filter into the filter holder. The specimens appear brigh, because they reflect the light from the microscope into the objective.

When to Use a Dark Field Microscope

Dark field microscopes 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 microscopy 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 microscopy Advantages and Disadvantages

No one system is perfect, and dark field microscopy may or may not appeal to you depending on your needs.

Some advantages of using a dark field microscope are:

Extremely simple to use

Inexpensive to set up (instructions on how to make your own dark field microscope are below)

Very effective in showing the details of live and unstained samples

Some of the disadvantages are:

Limited colors (certain colors will appear, but they're less accurate and most images will be just black and white)

Images can be difficult to interpret to those unfamiliar with dark field microscopy

Although surface details can be very apparent, the internal details of a specimen often don't stand out as much with a dark field setup.

Below are contrasting examples of dark field (left) versus bright field (right) illumination of lens tissue paper. Note how they both create a different style of image.

Dark field illumination Bright field illumination

Admit it, by now you're curious to check out your own dark field! You can create one with minimal time and effort. Just read on…

How to Make a Dark Field Microscope

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
Glue
Scissors
Pen

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.