Microscopy refers to method to observe/visualize the minute particles that are invisible to naked eyes. E.g. Bacteria, Viruses are the organisms that are not observed through our naked eyes. Visualization of them are only possible through Microscope. It is the process of microscopy that enables their visualization using microscope.
Microscopy is the technical field of using microscopes to view samples & objects that cannot be seen with the unaided eye. It is a simple, direct technique for examining the morphology of cells and their organelles.
A microscope is an instrument that magnifies objects otherwise too small to be seen, producing an image in which the object appears larger. Most photographs of cells are taken using a microscope, and these pictures can also be called micrographs.
- Microscope is an instrument, the magnification of which enables us to see microorganisms and their structures otherwise invisible to naked eyes.
- The magnifications attained by microscopes ranges from 100x to 400,000x.
On the basis of number of lens, Microscope can be classified as:
- Simple microscope- Have only one lens. E.g.. Magnifying glass
- Compound microscope- Have multiple lens. E.g. Light Microscope, Electron microscope.
Because of the way these lenses are arranged, compound microscopes can bend light to produce a much more magnified image than that of a magnifying glass.
- Magnificationis a measure of how much larger a microscope causes an object to appear. E.g. 500x magnification means 1 mm of an object is viewed as 500 mm magnified image under microscope.
- Resolutionof a microscope is the smallest distance by which two points can be separated and still be distinguished as separate objects. The smaller the resolution, higher the resolving power of the microscope and the better the clarity and detail of the image.
If a microscope has high magnification but low resolution, bigger version of a blurry image is obtained under microscopic observation. Both magnification and resolving power plays an important role in microscopy of the specimens.
Depending upon the principle on which magnification is based, microscopes are of two categories;
- Light or Optical microscope- It includes Bright-field, Dark-field, Fluorescence and Phase-contrast.
- Electron Microscope- It includes Transmission electron and Scanning electron microscope.
- Light Microscopy
In light microscopy, magnification is obtained by system of optical lenses using light waves.
In a light microscope, visible light passes through the specimen (the biological sample) and is bent through the lens system, allowing the user to see a magnified image. An advantage of light microscopy is that it can often be performed on living cells, so it’s possible to watch cells carrying out their normal behaviors (e.g., migrating or dividing) under the microscope.
In bright field light microscope, passes through the specimen and image is formed directly. The microscopic field is brightly lighted and microorganisms appear dark because they absorb some light.
Dark field microscope works under dark-field technique in which background is dark and objects are brilliantly illuminated. It consists of special condenser that transmits hollow cone of light from source of illumination. Microscopic field is dark as light directed towards condenser doesn’t enter objective lens.
Phase contrast microscope is based on principle that light passing through one material and into other material of slightly different refractive index and thickness will undergo change in phase. It can be used to observe living unstained cells.
In fluorescence microscopes, light passing through lens are absorbed by specimen and later it emits light which forms an image. E.g. confocal microscopy is type of fluorescence microscope. A confocal microscope uses a laser to excite a thin layer of the sample and collects only the emitted light coming from the target layer, producing a sharp image.
Fluorescent microscope uses ultraviolet light as its light source. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color.
- Electron Microscopy
Electron microscopes produce an image of a specimen by using a beam of electrons rather than a beam of light. As electrons have shorter wavelength than visible light, electron microscopes to produce higher-resolution images.
They are discovered to visualize the ultra-structure of microorganisms. It has magnitude of 10,000x or more. They can be used to visualize the subcellular structures of the cells. However, live cells cannot be visualized by electron microscopes as specimens are prepared through specialized fixation process which leads to cell death.
Electron microscope can be of two types, Scanning electron microscope and Transmission electron microscope.
In scanning electron microscopy (SEM), a beam of electrons moves back and forth across surface of a cell or tissue, creating a detailed image of 3D surface. Electron beam scans the surface of specimen which leads to release of secondary electrons and other types of radiation. SEMs are used in forensic laboratories to analyze a wide variety of samples, including paint, particles, fractures, toolmarks, and gunshot residue.
In transmission electron microscopy (TEM), sample is cut into extremely thin slices before imaging, and electron beam passes through the very thinly sectioned slice and projects the beam onto a specially treated plate that transmits the image to a monitor. TEM is often used to obtain detailed images of the internal structures of cells.