Dark field microscopy

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

Dark field microscopy (Dark Ground Microscopy) is an optical microscopy illumination technique used to enhance the contrast in unstained samples. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so 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

File:Dark Field Microscope.png

Diagram illustrating the light path through a dark field microscope.

1. Light enters the microscope for illumination of the sample.
2. A specially sized disc, the "patch stop" blocks some light from the light source, leaving an outer ring of illumination.
3. The condenser lens focuses the light towards the sample.
4. The light enters the sample. Most is directly transmitted, while some is scattered from the sample.
5. The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected.
6. Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.

Advantages and disadvantages

File:Mysis2kils.jpg

Dark field microscopy produces an image with a dark background.

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 are impressive.

The main limitation of dark field microscopy is the low light levels seen in the final image. This means the sample must be very strongly illuminated, and can cause damage to the sample.

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.

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Resources

• [2]
• Live blood photos under a Darkfield Microscope