Microscopy: Microscope is an instrument used to observe the objects which are not visible to our naked eye. Faber (1625) used the term microscope and it is derived from two Greek words namely,
Mikros= Small ; Skopian= To See.
History of Microscopy:
- During the 1″ century AD (year 100), glass had been invented and the Romans were looking through the glass and testing it.
- They tested with different shapes of clear glass and one of their samples was thick in the middle and thin on the edges.
- They discovered that if you held one of these “lenses” over an object, the object would look larger.
- It was observed that when the sun rays were focussed through the special glass lens it can start the fire.
- These early lenses were called magnifiers or burning glasses. The word lens is derived from the Latin word lentil, as they resembled the shape of a lentil beans.
- The early simple ‘microscopes” which were only magnifying glasses had one power, about 6X – 10X.
- These early magnifiers were called “flea glasses” as they were used to observe the fleas and other tiny insects.
- In 1590, two Dutch spectacle makers, Zaccharias Janssen and his father Hans started testing with the lenses.
- They placed several lenses in a tube and made a very significant discovery.
- The object near the end of the tube appeared to be greatly enlarged, much larger than any simple magnifying glass could achieve by itself.
- They invented the compound microscope which uses two or more lenses.
- These compound microscopes were later improved by Galileo (1610) and Robert Hooke (1660) Galileo added a focusing device to his microscope.
- Robert Hooke observed the microorganisms and documented them in his book “Micrographia” in 1965.
- Robert Hookeis called the “English Father of Microscopy” also spent his life working with microscopes and improved their design and capabilities.
- Antony van Leeuwenhoek (1632-1723) was skilled in art of glass blowing and fine metal work. He made small glass lenses with great curvatures by grinding and polishing.
- Hedevelopeda magnifying glass for trade purposes in 1653. He built a simple microscope during 1671 and started observing different objects like skin, hair, saliva, leaves, blood etc. He saw many creatures which he named”animalcules” which we now know as protozoa and bacteria. From his great contributions and many discoveries Antoni van Leeuwenhoek (1632-1723) is called the “Father of Microscopy”.
- The term ‘microscope’ is divided into two words, ‘micro’ means small or tiny and ‘scope’ means to observe. A microscope is defined as an optical instrument consisting of a lens or combination of lenses for making enlarged or magnified image of minute objects.
- Microorganisms are very small to be seen by naked eye. The human eye is unable to see an object less than 0.1 mm size placed at a distance of 25 cm.
- Therefore, it is necessary to magnify it so that it can be seen clearly and studied.
- There are two basic types of optical microscopes:
- Simple microscopes consist of only one lens or magnifying glass held in a frame which is adjustable and provides stand for holding the object to be viewed and a mirror for reflecting the light.
- Compound microscopes: It differs from a simple microscope in having two sets of lenses namely the objective lens and the ocular or eyepiece lens mounted in a holder called body.tube.
Principles of Microscopy
- Mechanical tube length: It is the length of the body tube of the microscope which is mostly 160 mm.
- Projection distance: It is the distance between the virtual image and the ocular or eyepiece lens which is usually 250 mm..
- Depth of focus: It is the depth of sharpness which depends on the numerical aperture (NA) and the magnification and is inversely proportional to both. Higher the magnification and NA lower is the depth of focus.
- Numerical aperture (NA): Numerical aperture is the light gathering capacity of the objective. The measure of the resolving power of an objective is the numerical aperture. The larger the NA the greater the resolving power of the objective and the final the detail it can reveal. NA is related to the refractive index (n) of a medium through which light passes. Therefore, in order to obtain higher working numerical apertures, the refractive index of the medium between the front lens of the objective and the specimen cover slip must be increased. (n = 1 for air; n = 1.51 for oil or glass). sin α is the sine of semi angle of the aperture and α is the half angle of the cone of light entering the objective lens from the specimen.
- NA = n sin α
- sin α cannot exceed the value of 1 and the refractive index of the optical instrument is constant. A higher refractive index (n) medium must be used. Oil immersion microscopes use oil to increase the refractive index and the optical resolving power of the microscope.
- In the figure light enters the object and radiates from it in all the directions. Some rays (C) do not enter the objective while some rays (D) get reflected from the coverslip Ray B forms an angle a with the axis.
- We can have a maximum angle of 90°. Air has refractive index of 1.0 and the sine a (90) equals to 1. So, NA comes to 1.
- If water is used as the medium NA is 1.30. With oil immersion NA is 1.50.
- Resolving Power: The resolving power of a compound microscope can be defined as the ability of the microscope to distinguish between two points lying very close to each other. The greater the resolving power, the smaller the minimum distance between two lines or points that can still be distinguished.
- Limit of resolution: It is defined as the minimum distance at which two objects lying very close to each other appear as two distinct objects. It can be calculated as Limit of resolution = 0.61 2/n sin a (NA)0.61 is the constant representing the minimum detectable difference in the contrast. 2. is the wavelength of light. NA is Numerical aperture (light gathering capacity).To achieve high resolution n sin a i.e. NA must be large. So, the objective lens is kept as close to the specimen as possible. The limit of resolution is inversely proportional to the resolving power. sin a can not exceed the value of 1 and the refractive index of the optical instrument is constant.
- Working distance: It is the distance between the specimen and the front lens of the objective when the specimen is in sharp focus. It varies with the type of the objective used (10X, 45X, 100X). As the magnification of the objective increases the working distance decreases.
- Magnification: Magnification is the process of enlarging the apparent size, not physical size, of something. Optical magnification is the ratio between the apparent size of an object (or its size in an image) and its true size, and thus it is a dimensionless number. Optical magnification is sometimes referred to as “power” (for example “10 x power”). The magnification of a microscope is given by MA = Mo x Me where, M, is the magnification of the objective and M. is the magnification of the eyepiece. The magnification of the objective depends on its focal length f, and on the distance d between objective back focal plane and the focal plane of the eyepiece (called the tube length). The magnification of the eyepiece depends upon its focal length fe.
Types of Microscopes:
Basically microscopes are classified into two main types namely:
1.Light microscopes
2. Electron microscope
- LIGHT MICROSCOPES: In these, light rays constitute the source of illumination
to focus the object or specimen. These include;
a. Simple microscope– It contains single lens system to magnify the specimen.
b. Compound microscope- It contains two lens systems to magnify the specimen. - ELECTRON MICROSCOPES: In these Electron beam constitute the source of illumination.
Light microscopes are of following types:
- Bright field microscope
- Dark field microscope
- Fluorescence microscope
- Phase contrast microscope
- Ultraviolet microscope
- Interference microscope
Electron microscopes are of 2 basic designs:
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
