A microscope is an optical tool that is used to magnify things that are too small to see with the naked eye. Have you ever tried using your naked eye to view the tiniest object or sample? We can’t see microorganisms with our naked eyes, but we can view them with microscopes. The microscope produces enlarged images of these micro-objects, allowing the user to examine minute features at a scale appropriate for research and analysis. Microscopy is the use of microscopes to examine things that are too tiny to see with the naked eye. This page will tell you about the history of microscopes, different types of microscopes, and how they are used.

Definition of Microscopy

It is derived from two Greek words, i.e. micros, which means small and scope, meaning to view or look at. Microscopy is the branch of science that deals with microscopes to view various cells and tissues invisible to naked eyes.

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History of Microscopy

1. The history of optical or light microscopy dates back to the 17th century.
2. Galileo Galilei and Cornelis Drebbel are considered early practitioners of the microscope.
3. Hans and Zacharias Janssen, Dutch spectacle makers, invented the first so-called compound microscope.
4. Antonie van Leeuwenhoek, in 1670, made the first high magnifying compound microscope and used it to observe live cells as well as microbes for the first time. He takes the credit of being called the ‘father of microscopy’, ‘father of microbiology’ and ‘father of biology’.
5. In 1733, an English optician, Chester Moor Hall discovered that a combination of a convex crown-glass lens and a concave flint-glass lens could help to correct chromatic aberration in a telescope.
6. In 1774, Benjamin Martin of London produced a pioneering set of colour-corrected lenses for a microscope.
With continuous improvements in optical glasses and lenses, optical microscopes were also improved. Now, the field of microscopy itself has become a branch of life sciences.

Terminologies Associated with Microscopy

1. Magnification is the measure of enlargement an object appears to be when looked at from the microscope.

2. Resolution: It can be defined as the shortest distance between two points distinguished as separate entities. The smaller the object, the higher the resolution of a microscope gives detailed images with better clarity.

3. Micrography: The field of taking photographs through microscopes is called micrography.

Formula to Calculate the Resolution Power of Microscope

The resolving power of a lens can be calculated using the following formula:

\(\varepsilon = 0.61 \times \frac{\lambda }{{{\rm{N}}{\rm{.A}}{\rm{.}}}}\) (Reyleigh formula)
\(\lambda :\)Wavelength
\({\rm{\lambda = 0}}{\rm{.55\mu m}}\) is used for visible light
N.A.: Objective lens N.A.

Light or Optical Microscopy

When the light source used to illuminate the field in a microscope is white light (visible light), it is called light microscopy. White light is transmitted or reflected through the biological specimen, and then the incoming light passes through a single or series of lenses to reach the observer. Light microscopy mainly uses two microscopes, i.e. simple and compound microscopes.

Simple Microscope

Definition: Simple microscopes are single-lens microscopes, sometimes also referred to as dissecting microscopes.

1. Lens used: It has a magnifying glass with a double convex lens with a short focal length.
2. Principle: A simple microscope works on the principle that when a tiny object is placed within the focal length of a convex lens, it gives a virtual, erect and magnified image.
3. Magnification: Magnification of a lens depends on its focal length and it is generally smaller than the compound microscope.

Diagram of Simple Microscope

Compound Microscope

Definition: Compound microscopes are dual-lens microscopes. The two lenses are called objective lens and eyepiece. It is commonly referred to as a student microscope.

1. Lens used: A compound microscope uses two convex lenses, one in the eyepiece and another in the objective. The eyepiece lenses vary with their focal lengths and hence the magnifications also differ. A student microscope comes with three objective lenses with magnifying power of \({\rm{10 x, 40 x}}\) and \({\rm{100x}}\) while magnifying power of the eyepiece can be \({\rm{5x, 10x, 15x, 20x}}\) and \({\rm{30x}}\)
2. Principle: The objective lens receives light scattered from the specimen, while the eyepiece is used for final observation of image. it gives a virtual, erect and magnified image.
3. Magnification: Magnification of a lens depends on its focal length and it is generally higher than the simple microscope.

Diagram of Compound Microscope

Parts of the Compound Microscope

A typical compound microscope has the following parts:

• 1. Head/Body: It is the upper part of the microscope. The body of the microscope holds the following parts:
  • (a) Eyepiece: It is also called an ocular lens. It is the lens present at the top end of the metal tube. Monocular models have a single tube, whereas binocular models have two tubes, each with one eyepiece whose distance can be adjusted.
  • (b) Eyepiece tube: This is a metal tube, \(160\,{\rm{mm}}\) (\(6.3\) inches) long. This length is based on the resolution of human eyes and is designed to minimise aberrations.
  • (c) Objective lens: These are the primary lenses on the microscope.
  • (d) Nosepiece: holds the objective lenses, has a revolving movement so that a particular objective can be selected to face the specimen.
  • (e) Coarse and Fine Focus Knobs: They are used to move the body tube to focus the image. Coarse knobs are used to do rough focusing of the image, while fine focus knobs are used to remove any blur focus and are completely based on the resolution power of the eye.
  • (f) Stage: This is a flat platform where a specimen is mounted. This is a mechanical stage, and slight movements are possible for better illumination.
  • (g) Stage clips: Hold the slide firmly in position, and finger movements are done to view different areas of the specimen.
  • (h) Aperture: The hole through which the transmitted light reaches the stage and, in turn, the specimen.
  • (i) Iris Diaphragm: Control the amount of light reaching the specimen. This is required as different specimens require a different amount of light to get sharp contrasts and view with better resolution. Thus, the iris diaphragm helps to get better contrast by controlling the amount of light.
  • (j) Condenser: It is located below the diaphragm and can be moved up and down to focus light on the specimen.
• 2. Base: This part supports the microscope, and holds the illuminator or the mirror.
  • (a) Illuminator: If present, is usually a low voltage halogen lamp. But many models also use natural light and are directed towards the aperture using a mirror.
• 3. Arm: this is the connecting part that connects the upper and lower parts of the microscope and microscopes are picked up or moved using the arm. Arm and base are joint with an inclination joint which has limited movement to adjust the illumination and light through the specimen.

Working Procedure of the Compound Microscope

The procedure to mount a specimen and observe it with a compound microscope goes as follows:
1. A biological specimen is mounted on a transparent glass slide immersed in glycerine or water and covered with a coverslip.
2. Such a ready slide is then mounted on the stage between the condenser and the objective lens.
3. A beam of visible light is focused on the specimen using a reflector mirror and condenser.
4. Iris aperture can also be adjusted to control the amount of light.
5. An objective lens (fixed on the revolving nosepiece) is chosen, depending on the magnification required. A \({\rm{100x}}\) objective lens requires an oil immersion on the specimen.
6. The objective lens then picks up the light through the specimen and an observer can view the image through the eyepiece
7. Based on the magnification and resolution of the image, a coarse and fine adjustment knob can be used to get a clear image.
8. If oil immersion is used, then the objective lens, specimen, and the stage have to be wiped clean with a cotton cloth.

Difference Between Simple and Compound Microscope

	Simple Microscope	Compound Microscope
\(1\)	Uses a single lens	Uses two-lens system
\(2\)	Only one lens is present	\(3\) to \(5\) objectives are present and one or two eyepiece lens are present
\(3\)	Magnification is limited to the single-lens used	Magnification is the multiplication of magnification of the eyepiece and the objective lens
\(4\)	The condenser lens is absent	A condenser lens is used to control the intensity of light entering the specimen
\(5\)	Coarse adjustments is very limited while the fine adjustment is not possible	Coarse and fine adjustments for the clear image are good and done with a coaxial knob
\(6\)	The light source is natural	The light source can be natural or artificial (halogen bulbs)
\(7\)	The mirror is concave, reflecting type	The mirror is plane at one side and concaves on other side

Techniques of Light Microscopy

Light microscopy or optical microscopy has many techniques developed based on the contrast required or the part of the specimen to be highlighted. All these variants are designed for specific purposes. A few of them are:

Brightfield microscopy: The common student microscope or light microscope is called a brightfield microscope because the image is produced against a brightly illuminated field.

• 1. The specimen is denser and somewhat opaque than the surroundings.
• 2. Light passing through such a specimen is absorbed.
• 3. Brightfield microscopy is used to observe preserved, stained or even live biological specimens.
• 4. The advantages of this simple type of microscopy are ease to handle and minimum sample preparation.
• 5. Disadvantage is that resolution remains low.

Darkfield microscopy: In optical microscopy, the dark field technique is used to enhance the contrast in an unstained specimen.

• 1. The directly transmitted light is minimised and instead, light scattered by the specimen is used to form the image.
• 2. This produces the classic dark, almost black backgrounds with bright objects on it and hence the name dark field microscopy.
• 3. It can be used to observe live specimens.

Phase-contrast microscopy: In biological specimens, there is a slight difference between the refractive index (R.I) of the cell or parts of the cell and the surrounding medium, which limits its focus and resolution. This difference is utilized in phase-contrast microscopes.

• 1. The phase differences between the light diffracted by the specimen and the undiffracted light from surroundings are utilized to create brightness variations.
• 2. This is particularly used to view cell structures, like a nucleus, which are otherwise invisible in bright field microscopy.
• 3. The phase-contrast microscope made it possible for biologists to study living cells, like cells during cell division.

Interference microscopy: Interference microscopy uses a prism to split light into two slightly diverging beams that then pass through the specimen.

• 1. It is thus based on measuring the differences in refractive index upon recombining the two beams.
• 2. Interference occurs when a light beam is retarded or advanced relative to the other.
• 3. Interference microscopy is superior to phase-contrast microscopy.

Fluorescence microscopy: This type of microscopy uses the specimen/samples that fluoresce.

• 1. Light of one wavelength is used to illuminate the sample and the light emitted from the sample is used to create an image.
• 2. The cells or parts of cells are labelled with fluorescent dyes.
• 3. This method is used in modern life science studies as it is highly sensitive allowing detection of even single molecules.

Polarizing microscopy: These are conventional microscopes but have an additional feature to use polarizing light.

• 1. A polarizing filter is present which allows the light to be polarized and then illuminate the object.
• 2. If the object is birefringent, it splits the light into two beams with different polarizations.
• 3. The filter fitted below the eyepiece blocks all the polarized light except one.
• 4. The analyzer can be rotated to get maximum contrast for a better image.
• 5. This type of microscopy is used to determine the orientation of native molecular structures in cells and to analyze the early developmental stages of organisms

Other Types of Microscope

1. Electron Microscope: The basic difference between a light microscope and an electron microscope is that an electron microscope uses a beam of electrons rather than visible light. Electrons have much shorter wavelengths than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.

2. Subcellular structures and their details can be clearly observed.

3. The major disadvantage of electron microscopes is that live cells cannot be observed as the samples have to be prepared through an extensive fixation process.

4. Electron microscopes are of two major types-scanning electron microscope (SEM) and transmission electron microscope

(a) Scanning Electron Microscope (SEM): a beam of electrons moves back and forth the surface of a cell or tissue and gives a very nice \({\rm{3D}}\) view. The best resolution of SEM in \(2011\) was \(0.4\,{\rm{nm}}\)

(b)Transmission Electron Microscope (TEM): the specimen is cut into very thin slices and the beam of electrons runs through the specimen. TEM is used to know the structures of cellular organelles.

5. Electron microscopes are heavier, bulkier, and very expensive than light microscopes. Also, specimen preparation is an extensive and time-consuming process.

Ultraviolet (UV) microscopes: It uses ultraviolet light to illuminate specimens. Ultraviolet radiations have much shorter wavelengths than visible light to obtain high-resolution images. This type of microscopy is useful, particularly for knowing the growth of protein crystals.

Infrared microscopes: The microscopy performed using infrared wavelengths is referred to as infrared microscopy. It is widely used in research, industry and one of the applications is in the forensics of civil and criminal cases. It is used to detect if two samples are the same or not.

Confocal laser microscopes: Laser illumination sources are used in various types of microscopes. This is an emerging field. Confocal microscopy is used where \({\rm{3D}}\) structures are important.

Significance of Microscopy

The importance of microscopy is as follows:

• 1. Microscopy has vast applications in biology and medicine.
• 2. It is primarily used to know the structures at tissue and cellular levels.
• 3. Subcellular structures are also explored using various types of microscopes.
• 4. Infrared microscopes are used to detect if two samples are the same or not.
• 5. UV microscopy is useful particularly for knowing the growth of protein crystals.
• 6. Dark field microscopy is useful to observe live specimens.
• 7. Confocal microscopy is used where \({\rm{3D}}\) structures are important.
• 8. Fluorescence microscopy is used to detect signals at a single-molecule level.

Summary

In today’s scientific world, a microscope is a commonly used instrument in biological sciences and medicine. It is used from observing simple unicellular organisms, various biological tissues to very high-resolution subcellular structures. Applications of microscopy in biological sciences and medicine are enormous, and the very field itself is engaging and mesmerizing.

Recent advances in fluorescence microscopy for contrast and sensitivity are such that scientists can detect signals at a single-molecule level. Although heavier and expensive, electron microscopes are needed in high throughput studies where extreme levels of magnifications are required. Molecular imaging and cell imaging have become the new buzzwords. New advancements in histological techniques, commonly used dyes and stains, and the development of various fluorochromes are enabling scientists to explore the living world around us with new magnifications.

FAQs

Q.1. What are the types of microscopes?
Ans: Various types of microscopes are used in the sciences. A compound light microscope is the most common. Light microscopes, electron microscopes, confocal laser microscopes are a few other examples. 

Q.2. What is microscopy?
Ans: Microscopy is the branch of science that uses various types of microscopes to see objects that are otherwise invisible to naked eyes.

Q.3. What is the principle of a simple microscope?
Ans: A simple microscope uses the principle that when an object is placed within its focal length, a virtual, erect and magnified image of the object is formed.

Q.4. What is microscopy used for?
Ans: Microscopy has vast applications in biology and medicine. It is primarily used to know the structures at tissue and cellular levels. Subcellular structures are also explored using various types of microscopes.

Q.5. Which is the most common microscope used in laboratories?
Ans: Compound microscope also called student microscope is the most common microscope used in laboratories. 

Q.6. What are the two main types of microscopes?
Ans: Simple single-lens microscopes and compound or double lens microscopes are the two main types of light microscopes.

Q.7. What are the \(14\) parts of a microscope?
Ans: The following image shows all the parts of a compound microscope.

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