Microscopy: Principles, Techniques, and Applications in Microbiology

Microscopy in Microbiology

General Principles of Microscopy

Microscopy is a fundamental technique in microbiology, allowing scientists to observe microorganisms and their structures. Understanding the principles of microscopy is essential for interpreting microscopic images and conducting laboratory work.

• Wavelength of Radiation: The distance between two consecutive peaks of a wave. Shorter wavelengths provide higher resolution, allowing finer details to be observed.

• Magnification: The process of enlarging the appearance of an object using lenses. Magnification is achieved through the refraction (bending) of light as it passes through glass lenses.

• Resolution: The ability to distinguish two points that are close together as separate entities. Higher resolution means better clarity and detail in the image.

• Contrast: The difference in intensity between an object and its background or between different parts of an object. Contrast is crucial for distinguishing structures within a specimen.

Metric Units of Length

Microbiology involves measuring extremely small objects, so understanding metric units is essential. The metric system uses prefixes to denote different orders of magnitude.

Prefix	Meaning of Prefix	Metric Equivalent	U.S. Equivalent	Representative Microbiological Application
Meter (m)	—	1 m	39.37 in (about a yard)	Length of tapeworm, 7 m (e.g., 1.8–8.0 m)
Decimeter (dm)	1/10	0.1 m = 10-1 m	3.94 in	—
Centimeter (cm)	1/100	0.01 m = 10-2 m	0.39 in; 1 in = 2.54 cm	Diameter of a mushroom cap
Millimeter (mm)	1/1,000	0.001 m = 10-3 m	—	Diameter of white blood cell
Micrometer (μm)	1/1,000,000	0.000001 m = 10-6 m	—	Diameter of bacterial cell
Nanometer (nm)	1/1,000,000,000	0.000000001 m = 10-9 m	—	Diameter of a poliovirus

The Electromagnetic Spectrum

The electromagnetic spectrum encompasses all types of electromagnetic radiation, including visible light, ultraviolet (UV) light, X-rays, and more. The wavelength of light used in microscopy affects the resolving power of the microscope.

• Visible Light: The portion of the spectrum detectable by the human eye, used in light microscopy.

• UV Light: Shorter wavelength than visible light, used in fluorescence microscopy for higher resolution.

• Resolving Power: Increases as wavelength decreases. Electron microscopes use electron beams with much shorter wavelengths than visible light, providing much higher resolution.

Example: Electron microscopes can resolve structures as small as 0.2 nm, while light microscopes are limited to about 200 nm.

Magnification and Refraction

Magnification in microscopy is achieved by bending (refracting) light as it passes through lenses. Convex lenses focus light to a point, creating an enlarged, inverted image of the specimen.

• Refraction: The bending of light as it passes from one medium to another (e.g., air to glass).

• Convex Lens: Used in microscopes to focus light and magnify the image.

Example: A simple microscope, like those used by Leeuwenhoek, uses a single convex lens to magnify microorganisms.

Resolution

Resolution is a critical property of microscopes, determining the clarity and detail of the image.

• Definition: The ability to distinguish two points that are close together as separate entities.

• Importance: Higher resolution allows for better visualization of fine details in microorganisms.

• Formula: The resolving power ($d$) of a microscope can be calculated as: $d = \frac{0.61 \lambda}{n \sin \theta}$ where $\lambda$ is the wavelength of light, $n$ is the refractive index, and $\theta$ is the half-angle of the maximum cone of light that can enter the lens.

Example: Electron microscopes have much higher resolution than light microscopes due to the shorter wavelength of electrons.

Limits of Resolution and Types of Microscopes

Different microscopes have varying limits of resolution, affecting the types of specimens that can be observed.

• Compound Light Microscope: Resolves objects down to about 200 nm.

• Transmission Electron Microscope (TEM): Resolves objects as small as 0.078 nm.

• Scanning Electron Microscope (SEM): Provides detailed surface images at high resolution.

• Atomic Force Microscope (AFM): Can resolve features at the atomic level (1 nm).

Example: Bacteria and most organelles can be seen with a light microscope, while viruses and large molecules require electron microscopy.

Contrast

Contrast is essential for distinguishing structures within a specimen. Without sufficient contrast, even high-resolution images may be difficult to interpret.

• Definition: The difference in intensity between two objects or between an object and its background.

• Importance: Critical for determining resolution and visualizing details.

• Methods to Increase Contrast:

  • Staining specimens with dyes

  • Using phase-contrast or differential interference contrast microscopy

  • Adjusting the light source or using filters

Example: Staining bacterial cells with crystal violet increases contrast, making them easier to see under the microscope.