Microscopy and Properties of Microscope

A microscope is an optical instrument that is used for magnifying objects too small to be seen by naked eye.

Investigations or studies of cell architecture by means of the microscope are called microscopy, and the person who pursuing the study is called microscopist.

History of Microscopy:

1590:

The two Janssen brothers of Holland, Francis Janssen and Zacharias Janssen, who were spectacle makers built the first operational light microscope.

1611:

Kepler built the first compound microscope.

1665:

Robert Hooke developed the first laboratory microscope which has a magnification of 14-42 X. He observed small pores in sections of cork that he called cells.

1674:

Leeuwenhoek discovered protozoa by his self built microscope with magnification of 270 X. He discovered bacteria for the first time 9 years later.

1905:

Zsigmondy invented dark-field microscopy.

Properties of Microscope:

A microscope has dual property i.e. magnification and resolution. The usefulness of a microscope depends not so much on the degree of magnification but rather on the resolution. Resolution has nothing to do with the magnification.

(a) Magnification:

Magnification or magnifying power of a microscope is the degree of increase in size of optical image over the actual size of object being viewed.

Magnification = Size of retinal image seen with microscope/Size of retinal image with naked eye

Magnification of microscope is calculated by multiplying the magnification of the objective lens with that of the eye piece (ocular lens). For example, the magnification of eye piece is 10X and the magnification of objective lens is 40X, then the microscope magnifies the object by 10×40= 400 times i.e. magnification is 400X.

The human eye has no power of magnification, so magnifying glasses maybe used to magnify images up to about 10 times. A light microscope in which combination of lens used has a magnification of 100-2000 X. For higher magnification over400X, oil immersion lens can be used in which cedar wood oil placed between objective and the coverslip increase the light gathering properties of the lens.

Units of Measurement used in Microscopy:

1 metre (m) = 10² cm = 10³ mm = 10⁶ mm = 10⁹ nm = 10¹⁰ A

1 centimeter (cm) = 1/100 metre (m) = 0.4 inch

1 millimetre (mm) =1/1000 metre = 0.001 m – 10-³ m = 10-³ mm = 10⁶ nm = 10⁷ A 1 micrometre (mm)** = 1 /1000 mm = 0.001mm = 10-³mm = 10-⁶ m = 10³ nm = 10⁴ A 1 nanometer (nm) = 1/1000 mm = 0.001mm = 10-³ mm = 10-⁶ mm = 10-⁹ m = 10 A

1 angstrom (A)⁺ = 1/10 nm = 0.1 nm = 10^-1 nm = 10-⁷mm = 10-¹⁰m

Micrometers were formerly known as microns (µ), and nanometers as millimicrons (mµ).

The Angstrom is not an accepted measurement in the International system of Units. It is included here, however, because it was widely used in microscopey in the past.

(b) Resolution or Resolving power:

Resolution (= resolving power or resolving limit) of an optical device (eye or microscope) is its ability to distinguish between two very closely placed objects as separate objects. The resolving power of a microscope depends on (i) Wavelength of light (A) and (it) numerical-aperture (NA) of the lens system used. Resolution of a microscope can be calculated by Abbe Equation, after the name of German Physicist Ernst Abbe in 1876.

Resolution = 0.61λ/NA= 0.61λ/N Sinθ

0.61 = a trigonometric constant

λ = Wavelength of light used; 450-750 mm for visible light used in compound microscope, Blue light has shortest wavelength (λ =450nm) gives maximum resolution. Therefore, blue filter blue light commonly used in microscopy.

NA – N Sin θ; where N is the refractive index of the medium (usually air or oil) between the specimen and objective lens. For air N = 1.0 and for immersion oil N = 1.5.

θ or a- half angle of the cone of light entering the objective lens from the specimen. The maximum value of 6 for the best objective lens is 70° (Sin 70° = 0.94). The resolution of light microscope, using air and blue light, will be

Lm= 0.61x 450nm/ 1.0x 0.94= 292nm or – 0.3µm

If oil and blue light used, then Lm = 0.61x 450nm/ 1.0x 0.94= 194nmor – 0.2 µm

Thus, light microscope can never resolve two closer particles less than about 0.2 nm apart, no matter how many times the image is magnified. The resolution of electron microscope is about 0.0005 µm whereas the human eye is about l00 µm. It should be noted that lower the value of Lm Higher will be resolution, which can be done by changing A, N or 6. Resolution will increase with a decrease in A and with an increase in NA; i.e. Lm is inversely proportional to A and Lm is proportional to NA. The numerical aperture (NA) is the light collecting ability of lens.