Name: how light microscopes bring samples into view
Uploaded: 2023-04-30T11:59:33.000Z
Duration: 1 min 18 s
Channel: JoVE

Optical microscopes use light sources to illuminate samples. The light passes through a series of lenses; the condenser focuses the light on the sample, while the objective and ocular lenses create a magnified and inverted two-dimensional image.
The numerical aperture of a lens determines its ability to gather light. The amount of light gathered depends on the angle of the light cone and the refractive index of the medium the light passes through, for example,&#160; air, water, or oil.
Optical microscopes have a resolution limit of 0.2 micrometers. They can resolve prokaryotic cells or large organelles within eukaryotic cells, which are around zero point five micrometers in diameter.&#160;
The resolution of the objective lens is inversely proportional to the wavelength of light and the lens&#39;s numerical aperture; the smaller the value, the higher the resolution of the magnified image.
An optical microscope can produce an image with a maximum of 1000 times magnification, calculated by multiplying the magnifications of the objective and ocular lenses.

imaging biological samples with optical microscopy

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&#10799; and 10&#10799; objective lenses; the fine focusing knob is used for small-scale movements, especially with 40&#10799; or 100&#10799; 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&#10799;. 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&#10799; 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&#39;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.

Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in ...

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how light microscopes bring samples into view

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution.

A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The slide is then moved over the light with the x-y mechanical stage knobs. These knobs move the slide across the stage but do not lift it. After the specimen is centered over the light, the stage is raised or lowered to bring the image into focus.

The coarse focus knob is used for large adjustments with 4x and 10x objective lenses. The fine focus knob makes small adjustments, especially with 40x and 100x lenses. As images become more magnified, they also become dimmer because less light reaches each part of the image. For that reason, microscopes need strong lighting.

In a brightfield microscope, a high-intensity bulb below the stage acts as the illuminator. Light from the illuminator passes through the condenser lens, which focuses the light onto the specimen. The condenser can be set with the condenser focus knob. After the best distance is found, the condenser should not be moved just to change brightness. If less light is needed, the diaphragm between the condenser and the specimen can be opened or closed. In some microscopes, the rheostat, or dimmer switch, also changes the illuminator intensity.

A brightfield microscope forms an image because the specimen transmits, absorbs, reflects, or refracts light in different ways. The objective lens makes a real inverted image. The eyepiece, also called the ocular, then shows a magnified virtual image of that picture. Chromophores, which are pigments that absorb and reflect certain wavelengths of light, can affect how colors appear. Stains are often added to increase contrast and resolution. This makes structures in the sample appear darker than the bright background and allows sharp images at magnifications up to about 1000x.

Higher magnification does not keep improving resolution. It can make the image larger without making it clearer. This is why bacteria can be seen at about 400x, but smaller objects such as viruses cannot. At very high magnifications, resolution can drop because light passes through the thin air gap between the specimen and the lens. Air and glass have very different refractive indices, so air scatters light before it can be focused.

Oil immersion solves this problem. A drop of oil fills the space between the specimen and an oil immersion lens, which is designed to work with immersion oils. Because the oil has a refractive index very close to glass, it lets light leave the specimen at a wider angle and reach the lens more easily. This increases the amount of light collected and improves image resolution. Different oils can be used for different types of light.

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How Light Microscopes Bring Samples Into View | JoVE K12

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How Light Microscopes Bring Samples Into View

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