What is Microscopy?

Microscopy is a technical field of study about the use of microscopes to observe small objects like cells and micro-organisms that are not visible to the naked eye. A microscope is used in the field of microscopy, in order to see the object or organism that cannot be seen easily. Microscopes help us to observe the fine structure of a cell and its content.

Types of Microscope

Fundamentally, the microscopes can be divided into two categories: light microscope and electron microscope. But these two categories can further be divided into several parts according to their working principle.

Light Microscopy

In light microscopy or optical microscopy, beams of visible light are used to visualize a specimen under the study. The microscopes used in this microscopy are made with several glass lenses to magnify the image of the specimen. There are different types of microscopes that act by using light:

Compound Microscopy

A modern compound light microscope or bright field microscopy is commonly used to observe cells as a specimen, where the sample appears in color according to the color of the stain with a bright surrounding. Its body can be divided into main five parts. An illuminator or light source, which provides light to the specimen to be observed, a condenser having a variable numerical aperture that allows a definite amount of light to be passed to the specimen. The next one is a stage where the specimen is fixed on a glass slide with appropriate staining. The most important part is the objective lenses which magnify the image and one or two ocular lenses or eyepiece through which we can observe the specimen. The working principle of a compound microscope is very simple. The illuminator provides the light beams that are passed through the condenser, which directs the finite number of light rays to the stained specimen on the stage. After that light passes through the sample to the objective lens, which magnifies the resolution of the image by the type of the lenses. e.g., 10X or 10 times, 40X or 100X.

Darkfield Microscopy

With this imaging technique, a special condenser is used in the microscope which contains an opaque disk. The disk eliminates most of the light from the center and the specimen receives only reflected light. As a result, the sample glows with a dark background. The main advantage of darkfield microscopy is that the sample can be observed without staining and for that, we can observe living cells also the imaging resolving power better than that of the bright field technique.

Phase-contrast Microscopy

Phase-contrast microscophy is a special type of microscopic imaging technique where living cells can be observed in three-dimensions (3D) without staining. It contains an annular or ring-shaped diaphragm that reflects and divides the light beams into different phases and travels through the specimen. As a consequence, combing different phases of light creates a 3D image of a living organism. This imaging technique also reveals the fine ultrastructure of the organism.

Differential Interference Contrast (DIC) Microscopy

This is a microscopy technique that provides contrast to the specimen's image which would have no contrast when viewed under bright field microscopy. DIC microscopy uses two light beams created by a prism to produce color in the image, otherwise, the process is the same as the previously mentioned process but with a higher resolution.

Fluorescence Microscopy

Fluorescence microscopy is one of the most advanced microscopic imaging processes available. Here, the sample is first stained with fluorochromes and then excited with a certain wavelength of light (commonly, ultraviolet (UV) light) inside the fluorescent microscope. The excited fluorochromes are then emitted at a higher wavelength of light than the visible spectrum in the microscope. The image of the sample appears as a glowing body with a dark background. This imaging process is mainly used in the studies of intracellular interactions, clinical identification of bacteria, where fluorophore-labeled antibodies are used to bind with the desire microbes and visualized them under the fluorescence microscope. This method is also called the immunofluorescence microscopy technique.

Confocal Microscopy

The confocal microscopic imaging process is the best process to construct a three-dimensional image of a cell. In this process, a confocal microscope is used which scans the fluorochrome-stained specimen understudy in every section and generates a 3D model of the specimen with the help of computer software. The analysis of intracellular contents of a cell as well as cell signaling is done by confocal microscopy. Small sections of the fluorochrome stained specimens are illuminated with blue light and due to fluorescence, a slice of a cell appears in the ocular lens of the microscope, which is further computerized to restructure the entire 3D model of the sample cell.

Two-photon Microscopy

In this process, two higher wavelengths of light (red light) are used instead of one in two-photon microscopy. As a result, we can examine particular cellular structures in the depth of 2 mm deep in tissue. The later process is the same as confocal microscopy. The resolution of the resultant image is very high, and we can examine cellular processes in real-time under the microscope.

Electron Microscopy

Unlike optical microscopy, this microscopy method uses electron beams instead of light to create magnifications up to 100,000 times the specimen's original size. In an electron microscope, glass lenses are replaced by magnetic lenses to focus the electron beams on the specimen because electrons cannot penetrate glass, and the eyepiece of the microscope is replaced by a fluorescent screen or a computer monitor. The resolution of the image is much higher than that of conventional light or optical microscope, we can even examine and analyze viruses with this microscopy method. It is mainly divided into two categories,

Transmission Electron Microscopy (TEM)

In TEM, the specimens are first cryopreserved and dried under the vacuum. Then cut with a microtome knife and stained with carbon-metallic stain to create a replica. At the top of the microscope, an electron gun generates a shower of electrons which are condensed by magnetic lenses and focused on the specimen placed on the metallic tray. The beams of electrons pass through the specimen and further condensed by another set of magnetic lenses to focus on a fluorescent screen. The internal molecules of the specimen like proteins in the plasma membrane or chromosomes or vacuoles, polyphosphate granules of bacteria can be easily observed. Computer programs can add colors to the image for better analysis. Intracellular analysis and observation of substances less than 2 μm can be done by using TEM.

Scanning Electron Microscopy (SEM)

The basic mode of action of the scanning electron microscope is the same as TEM. But there are few differences between SEM and TEM. In SEM the electron gun generates a shower of electrons called primary electrons, which are condensed and focused by magnetic lenses on the specimen. The interaction of primary electrons with the specimen releases secondary electrons from the surface of the specimen that is detected by the sensor present inside the microscope. The sensor then creates an electrical signal which is then amplified by an amplifier and displayed either in a fluorescent display or a computer monitor.

This ultra-magnified imaging process creates 3D imaging of the specimen with excellent resolution.

Formulas

Limit of resolution = 0.61λ / n x sin α

where,

• λ = Wavelength of light used to illuminate the specimen
• n = Refractive index of the medium
• α = Angular aperture

Context and Applications

This topic is significant in the professional exams for both undergraduate and postgraduate courses, especially for:

• B.Sc. in Microbiology
• M.Sc. in Microbiology
• M.Sc. in Biochemistry
• M.Sc. in Biotechnology

Related Concepts

• Freeze fracture method
• Staining
• Fixation