BASIC PRINCIPLES OF MICROSCOPY

Contents
1. Definition of microscopy 2. Microscope 3.Light 4.Properties of light 5.Interaction of light with matter a.Reflection b.Retardation c.Refraction d.Absorption e.Polarization f.Fluorescence 6.Lens 7.Defects of lens a.chromatic aberration b.Spherical aberration c.Astigmatism d.Coma 8.Resolution 9.Magnification

1. What is microscopy ? 2. It is the science of using microscopes to view samples and objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). 3. Microscope is an optical instrument which is used to : 1. visualise fine details in the structure of an object 2. provide a magnified image of an object 3. measure length , angles , areas ,etc in the object 4. LIGHT 5. Vibration in the ether ( a hypothetical substance that fills the space) 6. Human eyes respond to wavelengths from 730-760 nm in red/ infrared region to about 360-380 nm in violet 7. Highest senitivity at 550 nm in green region 8. PROPERTIES OF LIGHT 9. AMPLITUDE: it refers to the strength of the energy or brightness of the 10. WAVELENGTH: it determines color and is measured in nanometers. 11. FREQUENCY: the number of waves per second is referred to as frequency. light.

Interactions of light with Matter 1. 2. 3. 4. 5. 6. Reflection Retardation Refraction Absorption Polarization Fluorescence It is important to understand these interactions to control contrast and visibility under microscope Reflection : Reflection is the change in direction of a wavefront at an interface between two different media so that the wavefront returns into the medium from which it originated

If the surface is smooth, we get specular reflection If the surface is rough, we diffuse reflection In microscopy, reflection is mainly used for study of the surfaces of opaque objects

Retardation:

Refraction change in direction of a wave passing from one medium to another caused by its change in speed.

Refraction is the process by which glass lenses work in a microscope. Also it may occur b/w specimen & its surroundings or b/w different parts of same specimen. So it may hinder one or more image forming interactions b/w light and specimen.

Total Internal Refraction

Refractive Index : The ratio of velocity of light in air to velocity of light in any substance.

SNELLS LAW

Absorption: Object is made visible because it reduces the amplitude of the light passing through it. Majority of living cells and tissues are transparent , that is, they do not absorb light, and they have to be rendered absorbent by treating them with various dyes.

Change of phase

This principle is used in phase contrast microscopy