Introductory Microbiology

Numerical aperture

• Numerical aperture refers to the light gathering ability of lenses. The angle q subtended by the optical axis and the outermost rays still covered by the objective is the measure of the aperture of the objective. It is the half aperture angle. The magnitude of this angle is expressed as a sine value. The sine value of half aperture angle multiplied by the refractive index n of the medium filling the space between front lens and the cover slip gives the numerical aperture (NA). NA=n sin q.
• With dry objectives the value of n is 1 since 1 is the refractive index of air. When immersion oil is used as the medium n is 1.56 and if q is 580. Then NA = n sin q = 1.56 x sin 580 = 1.56 x 0.85 = 1.33. The degree to which microscope objectives can be altered to increase the NA is limited. The maximum NA for dry objective is less than 1.0 and oil immersion objectives have an NA value of slightly greater than 1.0 (1.2 to 1.4). The wavelength of light used in optical microscopes is also limited. The visible light range between 400nm (blue light) and 700 nm (red light), or 0.4 µm to 0.7 µm (abbreviation nm stands for nanometer and is equal to 0.001 µm or 10-9m).
• Thus it is apparent that the resolving power of the optical microscope is restricted by limiting values of NA and the wavelength of visible light.