The invention of dark field microscopy techniques has allowed scientists and researchers to study in detail samples that have been previously shrouded in mystery. The transparency or certain characteristics of these samples have made bright field illumination unable to capture their images with great clarity but thanks to dark field microscopy techniques, they are now extremely observable and providing a lot of much-needed answers to the world of science.

What is Dark Field Microscopy?
Dark field microscopy is not the “default” setup for microscopes. This honor belongs to bright field microscopy and which utilizes light coming from a microscope’s lamp source. Specimens or samples can then be seen because of their reactions to the speed of light being used and the path it takes. Of course, not all specimen project excellent images through the microscope; this greatly depends on the specimen’s opacity and refractive index. When a specimen’s almost or completely transparent and when its refractive index is too alike with the surrounding medium then it will not be able to create a clear image with bright field microscopy. And that’s when dark field microscopy comes in.

In dark field microscopy, the microscope’s condenser doesn’t use a cone of light to illuminate the sample. Rather, it uses a concave cone of light and projects it to the area surrounding the sample. Because of this, the sample will consequently produce a bright image against its dark backdrop.

There are specialty microscopes manufactured to produce exclusive dark field images. These are equipped with expensive condensers to create the best dark field images. Ordinary microscopes can nevertheless use dark field microscopy techniques as well by placing a stop below the substage condenser. A stop is a simple opaque object that can turn the beam of light from the microscope’s lamp source into a hollow cone of light necessary for dark field microscopy.

Dark Field Microscopy Techniques: Transmitted Dark Field Illumination and Reflected Dark Field Illumination
These are the two most commonly used dark field microscopy techniques used today. Which to use will depend on your objectives and the type of specimen you wish to observe.

Reflected Dark Field Illumination – The magnifying powers of a reflected light microscope is improved when dark field applications are used. In reflected dark field illumination, only peripheral rays of light are able to reach the microscope’s deflecting mirror because an opaque occluding disk is partially blocking the light path leading through the microscope’s vertical illuminator. The top surface of your sample must be positioned upright on the microscope stage and facing the objective.

The latest models of reflected light illuminators are often referred to as universal illuminators since the mere addition of accessories can easily enable the illuminator to switch from various modes of reflected light. Some models can even double as transmitted light illuminators – which is another dark field technique and will be discussed later on. High-end models are sometimes equipped with built-in optical features to provide better magnification.

There are various ways that reflected dark field illumination is put into use today. Among other things, it can produce erect image capability, a feature that allows the microscope or illuminator to capture non-reversed letters and produce infinity-corrected optical systems free from ghost images and astigmatism. Besides these, reflected dark field illumination is used in medical diagnostics, materials and cell sciences, the semiconductor industry, and in fluorescence microscopy.

Transmitted Dark Field Illumination – In this application, a dark field objective pair or condenser is used. The objective has a built-in iris diaphragm which can decrease the objective’s numerical aperture until it’s lower than the value of the concave cone of light coming out from the microscope’s condenser. In transmitted dark field illumination, a cardioid condenser is used to enable internal mirrors to produce a beam of light onto the surrounding area of the sample.

Specimens that are not easily visible with bright field microscopes have better chances of being seen with dark field illumination. Such samples are usually unstained and unable to absorb light and they typically include small insects, fibers, bones, hair, yeast, unstained bacteria, cells in tissue cultures, diatoms, protozoa, and microscopic living aquatic organisms. As for non-living samples, thin sections of ceramics and polymers, refractive index gradients, minerals, colloidal particles, chemical crystals, dust-count specimens, and porosity differences are all possible to study with a dark field microscope.

Users must exhibit care when handling specimens for observation with dark field microscopy techniques. Be extra careful when adjusting the amount of light used on the sample – although you need to use more than the usual amount of light on the specimen, going overboard could easily damage the specimen itself.