10+ Terms to be known in Microscopy

Terms to be known in Microscopy – Some important terms you must know to understand microscopy are discussed here in this article.

Terms to be known in Microscopy

Magnification

Magnification is the process of enlarging the appearance of an object so that it appears bigger than its actual size.

• Calculation: The total magnification of a microscope is the product of the magnification of the ocular lens (eyepiece) and the objective lens.
• Formula: Total Magnification= Magnification of Ocular Lens × Magnification of Objective Lens
• Example: If the ocular lens has a magnification of 10x and the objective lens is 40x, the total magnification is 400x.

Resolution (Resolving Power)

Resolution is the ability of a microscope to distinguish two closely spaced points as separate entities. The higher the resolution, the clearer the image detail.

• Key Factor: Numerical Aperture (NA), wavelength of light, and the quality of lenses influence resolution.
• Formula: d = λ / 2 × NAd
  • d: Distance between distinguishable points (smaller ddd means higher resolution)
  • λ: Wavelength of light used
  • NA: Numerical Aperture of the objective lens
• Diffraction Limit: The smallest detail that can be resolved is limited by the wavelength of light, typically around 200 nm for visible light.

Numerical Aperture (NA)

NA measures the light-gathering ability of an objective lens and its capacity to resolve fine details.

• Formula: NA=n×sin(θ)
  • n: Refractive index of the medium between the lens and the specimen (e.g., air, water, oil)
  • θ: Half-angle of the maximum cone of light that can enter the lens
• High NA: Provides better resolution and clearer images. Oil immersion lenses have higher NA (up to 1.4) because oil has a higher refractive index than air.

Also Read: History and Development of Microscopy

Working Distance

The working distance is the space between the objective lens and the specimen when the image is in focus.

• Higher Magnification Lenses: Shorter working distances.
• Low Magnification Lenses: Longer working distances, allowing more room for manipulating the specimen.
• Significance: Shorter working distances increase the risk of damaging the specimen or the lens, but they provide higher resolution.

Depth of Field

The depth of field is the thickness of the specimen that remains in focus at a particular magnification.

• High Magnification: Shallower depth of field, requiring precise focusing.
• Low Magnification: Greater depth of field, keeping more of the specimen in focus simultaneously.
• Application: In imaging thick samples, adjusting the focus at different levels helps in creating a clearer picture of the entire specimen (often done in confocal microscopy).

Field of View (FOV)

The field of view is the diameter of the area visible through the microscope lens.

• Inversely Proportional to Magnification: Higher magnification results in a smaller field of view.
• Utility: A larger field of view allows more of the specimen to be observed at once, useful for scanning slides.

Focus

It refers whether the image is well defined or blurry (out of focus). The focus can be adjusted through course and fine adjustment knobs of the microscope which will adjust the focal length to get clear image. The thickness of specimen, slide and coverslip also decide the focus of the image. (Thin specimens will have good focus).

Brightness

It refers how light or the dark the image is. Brightness of the image is depends on the illumination system and can be adjusted by changing the voltage of the lamp and by condenser diaphragm.

Contrast

Contrast refers to the difference in light intensity between the specimen and the background, making the specimen’s features more distinguishable.

• Staining: Adding dyes (e.g., Gram stain) to increase the contrast of microorganisms.
• Phase-Contrast Microscopy: Enhances contrast without staining by amplifying differences in refractive index.
• Fluorescence Microscopy: Uses fluorescent dyes that emit light upon excitation, providing high-contrast images.

Illumination and Light Path

• Köhler Illumination: A technique that provides even, high-contrast illumination across the field of view.
• Involves precise alignment of the microscope’s light source and condenser lens to focus light optimally on the specimen.
• Brightfield vs. Darkfield:
  • Brightfield Microscopy: Light passes directly through the specimen, and the image is formed by transmitted light.
  • Darkfield Microscopy: Uses oblique light to enhance the visibility of specimens that are transparent or have little contrast.

Lens Aberrations

Aberrations are imperfections in the image caused by optical defects in the lenses.

Types:

• Chromatic Aberration: Caused by different wavelengths of light refracting at different angles, leading to color fringing.
• Spherical Aberration: Occurs when light rays do not converge at the same point, resulting in a blurred image.

• Correction:
  • Achromatic Lenses: Reduce chromatic aberration by combining different types of glass.
  • Apochromatic Lenses: Further reduce chromatic and spherical aberrations for improved clarity.

Sample Preparation

• Importance: Proper preparation of the sample is critical for achieving good imaging quality.

• Techniques:
  • Fixation: Preserves the structure of cells or tissues (e.g., using formaldehyde).
  • Staining: Increases contrast and highlights specific features (e.g., Gram staining for bacteria).
  • Mounting: Involves placing the specimen on a slide with a coverslip for examination.

For More : A to Z of Microscopy terms