Microscopy

 

Microscopy is the science of using microscopes to observe objects that are too small to be seen with the naked eye. In microbiology, microscopy is essential for studying microorganisms such as bacteria, fungi, protozoa, and viruses.

Importance of Microscopy in Microbiology

–       Study of morphology (shape, size, arrangement)

–       Identification and classification of microorganisms

–       Observation of stained and unstained specimens

–       Understanding cell structure and function

Microscope-

"Micro" refers to tiny, and "scope" refers to view or look at. Microscopes are tools used to enlarge small objects so that they can be observed and studied. They range from a simple magnifying glass to an expensive electron microscope.

In the field of microbiology, the invention of the microscope is mainly credited to Antonie van Leeuwenhoek (1632–1723). He was the first person to observe and describe microorganisms (which he called “animalcules”). He used a simple microscope with very high magnification (about 200–300×). In 1674–1683, he observed bacteria, protozoa, yeast, and sperm cells.

Note- Zacharias Janssen (c. 1590) → Invented the compound microscope

Antonie van Leeuwenhoek → First to use the microscope to study microorganisms

Classification of microscopes.

Microscopes can be classified based on various properties.  Based on the lens system, microscopes are of two types.

Simple microscope	Compound microscope
1. It has a single lens system.	1. It has two or more lens system.
2. Example:-Magnifying glass.	2. Example:-Compound microscope basically used in the laboratory.
3. Reading small letters, simple observation	3. Studying microorganisms, cells
4.Magnification Low (up to ~10×)	4. High (40×–1000×)

Based on the source of illumination, there are two types of microscope:

Light Microscope	Electron Microscope
1.   It uses light as a source of illumination.	1.   It uses an electron or electron gun as a source of illumination.
2.   Lens used in a glass lens	2.   The lens used is an electromagnetic lens.
3.   Here, a vacuum is not needed.	3.   A vacuum must be created since electrons can travel only in a vacuum.
4.   Resolution is low compared to the electron microscope.	4.   Resolution is very high, capable of viewing particles of size 0.1 to 0.2 nanometers (nm).
5.   Image can be directly seen by the human eye.	5.   Since the human eye can’t see electrons. Electrons are converted into an amazing image by striking a fluorescent screen.

Types of Microscope

Bright Field Microscopy

Bright Field Microscopy is the most basic, commonly used light microscopy technique where light passes directly through a sample, producing a dark image on a bright, illuminated background. It is essential for observing stained, fixed, or high-contrast,, colored samples in biology and materials science.

· Imaging Principle: Transmitted white light passes through the specimen, and the image is formed by the absorption of light in denser, stained areas.

· Applications: Commonly used in microbiology for examining stained bacteria and in cell biology for tissue sections.

Magnification

The microscope produces an enlarged image of the object that is examined through it. This enlargement is known as its magnification and is measured in diameter. Eg: A magnifying lens which gives an image 36 times as large as that of the object is said to have a magnification of 36 diameter or 36X(x=times).

Magnification is the process of enlarging the apparent size of an object. It tells how many times the image of the object is larger than the actual object.

Total Magnification=Magnification of Objective Lens×Magnification of Eyepiece (Ocular Lens)

Example:

• Objective lens = 40×
• Eyepiece = 10×
• Objective lens = 40×

Total Magnification=40×10=400×

Note: High magnification without good resolution gives a blurry image.

Resolution

Resolution (or resolving power) is the ability of a microscope to distinguish two points that are very close together as separate entities. In other words, it measures the clarity or detail of the image.

The ability of a microscope to distinguish two closely spaced objects as separate and distinct entities is called resolution. Eg. The human eye has the resolving power of 0.25mm, which means that two dots placed 0.25mm apart can be distinguished as two dots. If the distance between the two dots is less than 0.25, only one dot will be seen.

•        Resolving Power (R.P.) = λ/2 NA where λ= wavelength of light , N.A=Numerical Aperture

or

Formula for Resolution (d):  d=λ/2 NA

Where:

• d= minimum distance between two distinguishable points (in meters or micrometers)
• λ (lambda) = wavelength of light used (usually 400–700 nm for visible light)
• NA = Numerical Aperture of the objective lens

Interpretation:

The formula RP = λ / (2 x NA) indicates that the resolving power is influenced by both the wavelength of light and the numerical aperture.

The resolving power is inversely proportional to the wavelength of light (λ). As the wavelength decreases, the resolving power increases, allowing for better separation of closely spaced objects.

The resolving power is directly proportional to the numerical aperture (NA). Higher numerical apertures lead to greater resolving power. A larger NA allows the lens to capture more diffracted light, contributing to improved resolution.

• Smaller d → better resolution → can see finer details
• Higher NA or shorter wavelength → better resolving power