Working Mechanism of Light Microscope

Microscope is an instrument that is used for the visualization of things that are too small to be seen from the naked eyes. A light microscope uses visible light. Different parts of a compound microscope work together so that the final image is magnified.

      Working mechanism   

For the magnification of an object, light projects through an opening on the stage. Then the projected light hits the object and enters the objective lens. An image is created which becomes an object for the ocular lens.  Then the ocular lens, re-magnifies the image. Total magnification is magnification by objective lens × magnification by ocular lens. 

Step 1: Illuminator provides the light

Step 2: Diaphragm controls the amount of light provided by the source

Step 3: Condenser focuses light through specimens by condensing light rays to the strong beam

Step 4: Light from condenser is projected where it hits the object in stage and enter the objective lens

Step 5: Objective lens then magnifies the specimens

Step 6: Body tube transmits the image from the objective lens to the ocular lens

Step 7: Ocular lens re-magnifies the image formed by the objective lens.

 Resolution (or resolving power) 

Resolution or Resolving power is the ability of a lens to distinguish two adjacent points as distinct and separate. The shorter the wavelength of light used in the instrument, the greater will be the resolution. The white light (wavelength of light used: 450-550 nm) used in a compound light microscope has a relatively long wavelength and cannot resolve structures smaller than about 0.2 µm.

The numerical aperture (NA) is the widest cone of light that can enter the lens. The minimum distance (d or resolution) between two objects that reveals them as separate entities is given by the Abbe equation.

Lambda (λ): wavelength of light used to illuminate the specimen.

n sinθ: the numerical aperture (NA)

The angular aperture θ is ½ the angle of the cone of light that enters a lens from a specimen.

                               d= λ/2nsinθ

= 0.5λ /nsinθ , n=refractive index

When d becomes smaller, the resolution increases, and finer detail can be identified in a specimen. To make d smaller, value of λ should be smaller and value of NA should be greater. Thus the greatest resolution is obtained with light of the shortest wavelength, and an objective with the maximum NA.

Why oil is used in 100x objective lens?

The angle of the cone of light that can enter a lens depends on the refractive index (n) of the medium in which the lens works, as well as upon the objective itself. The oil immersion lens (100x) is exceedingly narrow. Due to various reflection and refraction many light rays do not enter the objective lens.

Air is the medium between the objective and the slide (with specimen). The refractive index for air is 1.00. Since sinθ cannot be greater than 1, no lens working in the air can have a numerical aperture greater than 1.00 (NA=nsinθ=1×1=1). One approach to increase the numerical aperture greater than 1 is increasing the refractive index with immersion oil so that a higher resolution can be obtained.

Immersion oil is a  colorless transparent liquid. Oil and glass slide has same refractive index i.e 1.5. Light projected from the illuminator passes the oil without deviation. Then it is projected upwards to the objective lens. With the increased amount of light entering the objective, the NA and resolution of the object increase, and one can observe objects as small as bacteria.

References 

  • Madigan, M. T., Martinko, J. M., Stahl, D. A., & Clark, D. P. (2011). BROCK Biology of Microorganisms (13thedition). Benjamin Cumming.
  • Prescott, L. M. (2002). Microbiology (5th edition). The McGraw-Hill Companies.

Sushmita Baniya

Hello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.

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