Microscopy, in this era of technological advancement, is an important tool in modern biological studies.
Microscopes can be of following types-
- Optical microscopy o Bright field o Interference reflection microscopy o Oblique illumination o
Fluorescence o Dark field o Confocal
o Dispersion staining o Single plane illumination microscopy o Phase contrast o Light sheet fluorescence microscopy
o Differential interference contrast o Deconvolution
- Electron microscopy
- Scanning probe microscopy 5. Digital holographic microscopy
- Ultraviolet microscopy
- Digital pathology (virtual microscopy)
- Infrared microscopy
- Laser microscopy
In this laboratory, we will use bright field optical microscopes to see single cells and the chromosomes that lie within. The image quality will largely depend on your ability to prepare slides and to use the microscope properly.
Principles of Bright field microscopy:
The basic principle of compound microscopy is that by stacking lens together, it is possible to see increasingly smaller objects. Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The simplicity of the technique and the minimal sample preparation required are significant advantages.
Protocol for cytological microscopy:
Gently clean the surfaces of microscopes and the lenses using soft lens paper.
Place the appropriately prepared slide on the stage and let it be hold by stage clips.
Using coarse adjustment screw, set the stage in its bottom most position.
Observe the object using 4x eyepiece lens.
Using coarse adjustment screw, slowly move the stage upward until the object is clearly visible.
Using fine adjustment screw, set the object at its critical illumination.
Use the next higher nosepiece lens for further magnification of the object. Further fine adjustment may be needed to clearly see the cells.
Place the cell of interest at pointer by adjusting the stage controlling screws.
Figure: Different parts of a bright field optical microscope
1. Microscope is a very delicate, sophistical and expensive instrument. Please handle it gently with utmost care.
2.Use fine softened cotton cloth gently, if soft lens paper is unavailable (don’t use tissue papers), to clean the lenses and the body of the microscope to avoid any contact of stains/chemicals and damage/scrath to the lens.
3.While using 4x nosepiece, take special care so that the slide doesn’t come in contact with the nosepiece lens. It may break your slide, damage the nosepiece lens.
4.If needed, move the microscope, by holding (& lifting) the arms (body) of the microscope.