A microscope is a magnifying instrument used to visualize things that are too small to be seen by the naked eye. A light microscope uses visible light. The working mechanism of light microscope has different parts of a compound microscope working together to magnify the final image.
An objective lens collects light from the specimen and produces a magnified primary image of the specimen. The eyepiece enlarges this primary image, converting it into one that can enter the eye pupil.
Principle of Light Microscope
The working mechanism of light microscope includes projection of light through an opening on the stage. Then the projected light hits the specimen (object) and enters the objective lens (lens close to the object). An image is created, which becomes an object for the ocular lens (eyepiece). Then the ocular lens re-magnifies the image.
Total magnification is magnification by the objective lens × magnification by the ocular lens.
Example
| Objective magnfication | Eyepiece magnification | Total magnification |
| 10X | 10X | 100 diameters |
| 40X | 10X | 400 diameters |
| 100X | 10X | 1,000 diameters |
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 the condenser is projected where it hits the object in stage and enters 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 the resolution. The white light (wavelength of 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 Abbe equation gives the minimum distance (d or resolution) between two objects that reveal them as separate entities.
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, the value of λ should be smaller, and the value of NA should be greater. Thus the greatest resolution is obtained with the light of the shortest wavelength and an objective with the maximum NA.
Why oil is used in 100x objective lens?
When a beam of light passes from air into a glass, it is bent towards the normal, and when it passes back from glass to air, it is bent far away from the normal. This has little effect on low power objectives, but with high power lenses, this bending limits the amount of light that can enter the lens and affects the objective’s NA and consequently its resolving power.
The bending effect and its limitations on the objective lens can be avoided by replacing the air between the specimen and the lens with an oil that has the same optical properties as glass, i.e., immersion oil.
Immersion oil is a colorless transparent liquid. Oil and glass slide has the same refractive index, i.e., 1.5. Light projected from the illuminator passes the oil without deviation and is projected upwards to the objective lens.
With immersion oil, the light passes in a straight line from glass through the oil and back to glass as though it were passing through glass all the way. Thus oil helps to collect extra oblique light and provides better resolution and a brighter image. Some 50X objectives and all 100X objectives are immersed in oil to 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.