33.1: Imaging Biological Samples with Optical Microscopy
Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage (a platform). Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses.
When images are magnified, they become dimmer because there is less light per unit area of the image. Microscopes produce highly magnified images, therefore, require intense lighting. In a brightfield microscope, an illuminator provides this light, typically a high-intensity bulb below the stage. Light from the illuminator passes through the condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than-maximal light levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator.
A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. The objective lens forms a real inverted image of the specimen. The final image visualized in the eyepiece or ocular is a magnified virtual image of the one formed by the objective.
Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, increasing contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image but without increased resolution. This allows us to see objects as small as bacteria, visible at about 400⨯ or so, but not smaller objects such as viruses.
At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the significant difference between the refractive indices of air and glass; the air scatters the light rays before the lens can focus them. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the image's resolution. A variety of oils can be used for different types of light.
This text is adapted from Openstax, Microbiology 2e, Section 2.3: Instruments of Microscopy.