Question

8. With regard to microscopy: a. What are the different types and why are they useful? What is the magnification limit and resolving power of each? b. Explain the importance of resolution and know how it's calculated C. Describe the importance of magnification and contrast d. What are the ways to provide contrast? What are the specific stains, why is each used, and how do they work?

Answer 1

a)The types of microscopes are given below:

• Brightfield microscope: The basic microscope which uses the transmitted light to visualize the sample at high magnification. The magnification is around 1300X and the resolving power is 10^-3m- 10^-7m.
• Darkfield microscope: It is similar to a light microscope but the condenser in this microscope is modified in a way that the scattered light from the specimen is viewed against the dark. Used to view living cells. The magnification is around 1000X and the resolving power is 0.2000 micrometer.
• Phase-contrast microscope: This microscope uses the principle of the differences in the thickness and refractive index of the parts of the object being visualized, can be transformed into different light intensity. The magnification is around 1000X and the resolving power is 0.2000 micrometer.
• Fluorescent microscope: Used to visualize specimens that are tagged by a fluorescent dye The fluorescent dye absorbs a wavelength and emits another wavelength which is visualized using this microscope. The magnification is around 1000X and the resolving power is 0.2000 micrometer
• Electron microscope: This microscope using electrons for illuminating the specimen. It can be of two types TEM and SEM. It is used to visualize the ultrastructure of many biological systems. The magnification is around 200000X for TEM and 10000X for SEM and the resolving power is 0.2nm for TEM and 20nm for SEM

b)Resolution is the shortest distance between two points in the specimen which can be distinguished by the microscope. This is important as the resolving power increases we can visualize tinier objects. It is calculated as

Resolving power= wavelength of light/2*numerical aperture of the objective lens

c) The importance of magnification is that it helps to enlarge the specimens so that we can understand the finer details in it. The contrast in the microscope helps us to differentiate between the specimen and the adjacent background relative to the overall background intensity

d) Changing various apertures like field aperture and condenser aperture, Giving stains to the specimen, image processing in real-time, Giving any additional magnification for video detectors, etc.

Different stains used are

• Methylene blue: Stains the nucleus with blue color. This is a positive dye which bings to the negative DNA
• DAPI:  A fluorescent stain that fluoresces when bound to DNA. Used to visualize nucleus of the cells
• Coomassie blue:  Stains proteins. Gives a blue color.  They bind to proteins trough ionic bond and Van der Waals interactions
• Rhodamine: Used in fluorescence microscopy for staining proteins. Forms complex with mycolic acids in acid-fast cell walls