AIM: To study the various types of Microscopy 
                         
                        THEORY: 
                         
                        Microscope is the most commonly used piece of apparatus in the laboratory. 
                        It produces greatly enlarged images of minute objects. 
                         
                        LIGHT MICROSCOPE 
                         
                         A Light Microscope can be simple or a compound microscope. 
                         
                        [A] SIMPLE MICROSCOPE   This is a simple hand magnifying lens. The 
                        magnification power of hand lens is from 2x to 200x. 
                         
                        [B] COMPOUND MICROSCOPE   This has a battery of lenses which are 
                        fitted in a complex instrument. One type of lens remain near the object 
                        (objective lens) and another type of lens near the observer’s eye ( eye piece 
                        lens). The eye piece and objective lenses have different magnification. The 
                        compound microscope can be monocular having single eye piece or, 
                        binocular which has two eye pieces. The usual type of microscope used in 
                        clinical laboratories is called light microscope. 
                           A compound microscope has the following parts: 
                                   ▪  Stand 
                                   ▪  Body 
                                   ▪  Optical system 
                                   ▪  Light/ Illumination system 
                         
                        1. Stand 
                         
                        This is Horse-shoe shaped in monocular microscope. It gives stability to the 
                        microscope. Binocular microscopes have a variety of ergonomic shapes of 
                        stand. 
                         
                        2. Body 
                         
                        It consists of limb which arises from the joint with which microscope can be 
                        moved in comfortable position. The stand and limb carry the following: 
                           i.     Body tubes 
                          ii.     Stage 
                         iii.     Knobs for coarse and fine adjustment 
        
       Body Tubes 
        
       There are two tubes: external tube which carries at its lower end a revolving 
       nose piece having objective lenses of different magnification while internal 
       tube is draw tube which carries at its upper end eye pieces. 
        
        
       Stage 
        
       This is a metallic platform which accommodates glass slide having mounted 
       object over it to be seen. Stage is attached to the limb just below the level of 
       objectives. It has an aperture in its centre which permits the light to reach the 
       object. Slide on the stage can be moved horizontally or vertically by two 
       knobs attached to slide holder. Just below the stage is substage which 
       consists of condenser through which light is focused on the object. The 
       substage can be moved up or down. The substage has an iris diaphragm, 
       closing and opening of which controls the amount of light reaching the 
       object. 
        
       Knobs for Coarse and Fine Adjustments 
        
       For coarse and fine adjustments, knobs are provided on either side of the 
       body. Coarse adjustment has two bigger knobs, the movement of which 
       moves the body tubes with its lenses. Fine adjustment has two smaller knobs 
       either side of the body. The fine focus is graduated and by each division 
       objective moves by 0.002 mm. 
        
       3. Optical System  
        
       Optical System is comprised by different lenses which are fitted into a 
       microscope. It consists of eye piece, objectives and condensers. 
        
       Eye piece 
        
       In monocular microscope, there is one eye piece while binocular microscope 
       has two. Eye piece has two plano-convex lenses. Their magnification can be 
       5x, 10x, or 15x. 
        
       Objective 
         
        These are made of a battery of lenses with prisms incorporated in them. 
        Their magnification power is 4x, 10x, 40x and 100x. 
         
        Condenser 
         
        This is made up of two simple lenses and it condenses light on to the object. 
         
         
        4. Light/ Illumination system 
         
        For day light illumination, a mirror is fitted which is plane on one side and 
        concave on the other side. Plane mirror is used in sunlight while concave in 
        artificial light. Currently, most of the microscopes have in-built electrical 
        illumination varying from 20 to 100 watts. 
         
         
         
        MAGNIFICATION AND RESOLVING POWER OF LIGHT MICROSCOPE 
         
        Magnification power of the microscope is the degree of image enlargement. 
        It depends upon the following: 
          •  Length of optical tube 
          •  Magnifying power of objective  
          •  Magnifying power of eye piece 
                 With a fixed tube length of 160 mm in majority of standard 
        microscopes, the magnification power of the microscope is obtained by the 
        following: 
              Magnifying power of objective x Magnifying power of eye piece . 
         
        Resolving power represents the capacity of the optical system to produce 
        separate images of objects very close to each other. 
         
                  Resolving power (R) =   0.61 λ 
                                                           NA 
                  
                  Where λ is wavelength of incidental light; and  
                         NA is Numerical aperture of lens 
                         Resolving power of a standard light microscope is around 200nm. 
         
        HOW TO USE A LIGHT MICROSCOPE 
         
          1.   The microscope must be kept in a comfortable position. 
          2.   Appropriate illumination is obtained by adjusting the mirror or 
           intensity of light. 
          3.  When examining colorless objects, the condenser should be at the 
           lowest position and iris diaphragm closed or partially closed. 
          4.  When using oil immersion, 100x objectives should dip in oil. 
          5.  After using oil immersion clean the lens of the objective should be 
           cleaned with tissue paper or soft cloth. 
         
        OTHER TYPES OF MICROSCOPY 
         
        DARK GROUND ILLUMINATION (DGI) 
         
        This method is used for examination of unstained living micro-organisms 
        e.g. Treponema pallidum. 
         
        Principle 
         
        The micro-organisms are illuminated by an oblique ray of light which does 
        not pass through the micro-organism. The condenser is blackened in the 
        centre and light passes through its periphery illuminating the living micro-
        organism on a glass slide. 
         
         
        POLARISING MICROSCOPE 
        This method is used for demonstration of birefringence e.g. amyloid, foreign 
        body, hair etc. 
         
        Principle 
         
        The light is made plane polarized. Two discs made up of prism are placed in 
        the path of light, one below the object known as polarizer and another placed 
        in the body tube which is known as analyzer. Polarizer sieves out ordinary 
        light rays vibrating in all directions allowing light waves of one orientation 
        to pass through. The lower disc (polarizer) is rotated to make the light plane 
        polarized. During rotation, when analyzer comes perpendicular to polarizer, 
        all light rays are canceled or extinguished. Birefringent objects rotate the 
        light rays and therefore appear bright in a dark background.