Question

Question 1) Microscopy. Give realistic examples for (i) standard light microscopy, (ii)

immunofluorescence microscopy, and (iii) electron microscopy on how these three distinct forms of

microscopy can be used to examine biological questions in cell and molecular biology. Indicate

limitations for these techniques, including if living cells or tissue or fixed (dead) samples are involved,

what resolution is reasonably possible (smallest object that can be seen) and if special reagents such as

antibodies are needed.

Answer 1

Standard light microscopy-

Schleiden and Schwann, used the standerd light microscopeto describe individual cells as the fundamental unit of life. The microscope, has several lenses which are used to magnify the image of a specimen under study. The total magnification is a product of the magnification of the individual lenses which is if the objective lens magnifies 100-fold along with the eyepiece which helps magnify 10-fold, thus, the final magnification recorded by the humans or on film will be 1000-fold.  Also, the most important property of the microscope is not just its magnification but is its resolving power, or resolution. It is defined as the ability to distinguish between two obejects which are very closely positioned togetherJust enlarging the image of a might make it blurry. The resolution is numerically equivalent to D, the minimum distance calculated between two distinguishable objects. Hence,they are inversely proportional. The smaller the D, better is the resolution. D is dependent on three parameters, the angular aperture, α, the refractive index, N, and the wavelength, λ, of light: D = (0.61λ) ÷ (N × sin α). Either by increasinng N or by decreasing the value of λ there will be better resolution.

Immunofluorescence microscopy-

There are 4 very useful dyes which are used for fluorescent staining. They are rhodamine and Texas red which will emit red light; Cy3, which emits orange light; and fluorescein, which emits green light. These above dyes have a low, but nonspecific affinity for biological molecules, They can be however be chemically coupled with purified antibodies which are specific for most macromolecule. When this fluorescent dye forms an antibody complex, this is added to a permeabilized cell or tissue section where the complex binds to antigens, which lightenes up when illuminated by the exciting wavelength. This is called immunofluorescence microscopy.

Electron microscopy-

The Electron Microscopy directs a beam of electrons through a biological specimen. Electrons are emitted by a tungsten cathode when it is heated electrically where the electric potential of the cathode is maintained at 50,000 – 100,000 volts. and the electric potential of the anode, near the top of the tube, is null/0. This sudden drop in voltage makes the electrons increases as they move toward the anode. There is a condenser lens which focuses the electron beam onto the sample; objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen or a piece of photographic film