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Stereo Microscope Primer: Transmitted Light Observation Methods

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Stereo microscope observation methods

Stereo microscopes are useful tools for examining specimens macroscopically and stereoscopically. These microscopes provide individual observation pathways for each eye, which gives the specimen some depth so that it appears as if you were examining it by eye.

Stereo microscopes offer two main types of illumination: reflected illumination and transmitted illumination.

  • Reflected illumination shines light downward and onto the specimen, enabling you to observe the reflection. This illumination type works best for opaque specimens, such as rocks, minerals, plants, insects, and ceramics.

  • Transmitted illumination shines light upward and through the specimen. This illumination type works best with specimens that are translucent, such as cells, tissues, embryos, zebrafish, or other small aquatic samples.

Stereo microscope

The specimen type determines which illumination will work best. In many cases, you may need to use multiple methods. Each illumination type also has several observation methods, as shown in Table 1 below.

Table 1 – Commonly Used Observation Methods for Stereo Microscopes

Observation Methods
Reflected Illumination Oblique
Coaxial
Polarized
Fluorescence
Transmitted Illumination Brightfield
Darkfield
Oblique
Polarized

Today, I’ll shine a light on transmitted illumination, so you can learn more about the observation methods within it.

4 Common Observation Methods for Transmitted Illumination

Now that we’ve covered the most commonly used observation methods for transmitted light, let’s explore their features and common applications.

  • Brightfield: Brightfield transmitted illumination is the most widely used method. Transmitted light is applied directly below the specimen. As light passes through the specimen, contrast is created by the attenuation of transmitted light through dense areas of the sample. If the sample isn’t stained and has no inherent color, then only small amounts of information can be obtained. In this case, it might be helpful to use other observation methods.

  • Darkfield: Darkfield transmitted illumination works well for samples that are unstained and transparent. The central light that typically passes through the specimen is blocked out, and only the oblique rays from every azimuth strike the specimen. These oblique rays are diffracted, reflected, and/or refracted by optical discontinuities. The result is a bright specimen on a black background.

  • Oblique Contrast: Oblique transmitted illumination also works well with unstained and transparent samples. This method enables you to adjust the angle of light to gain some contrast. It provides higher contrast compared to brightfield, but lower contrast compared to darkfield. With the oblique method, you can easily change the sample appearance to find the correct angle for illumination.

  • Polarized Illumination: Polarized transmitted illumination can be used on anisotropic specimens, which usually have multiple refractive indices (birefringent specimens). Polarization requires more equipment because the birefringent specimen must be placed between a polarizer and analyzer. The contrast comes from the interaction of plane-polarized light with the birefringent specimen, which will produce two separate wave components that are polarized in mutually perpendicular planes.

Brightfield observation of zebrafish

Zebrafish captured using brightfield observation

Oblique observation of medaka (left). Darkfield observation of medaka (right).

Medaka captured using oblique (left) and darkfield (right) observation methods

Expand the Scope of Observation

The ability to quickly and easily switch between observation methods can help you efficiently obtain information about your sample. Our LED transmitted-light stereo bases can help you switch between brightfield, oblique, darkfield, and polarized illumination—as well as different contrast methods—quickly and easily. Make sure to read our next blog post to see how!

Related Content

Brochure: Quad Position LED Transmitted Light Illumination Base

Video: Stereo Microscope Ergonomics

Brochure: Research Stereo Microscope System

Product Manager

Janeen Manning is a Product Manager for clinical and educational microscopes in the Scientific Solutions Group at Olympus Corporation of the Americas. She has a Bachelor of Science in biochemistry, microbiology, and molecular biology from the University of Maine and a Master of Liberal Arts in biotechnology from Harvard University. She has been with Olympus since 2017; prior to Olympus she worked for a medical device company specializing in infectious diseases.

Mar 09 2020
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