1.2. Numerical aperture

The numerical aperture of a microscope objective is defined as a measure to gather light and, thus, resolve fine specimen detail at a fixed objective distance.

The numerical aperture is given by the following formula:

 

NA = n*(sinμ)

where,

• n refers to the refractive index of the medium between the specimen's cover glass and the front lens of the objective. It is, n = 1.00 for air and n = 1.5 for immersion oil.

μ is one half the angular aperture (cone angle). The bigger the value of μ, the higher is the numerical aperture.

 

The numerical aperture of a lens depends upon two parameters, the angle of incidence of light onto the lens, and the refractive index (n) of the glass of which the lens is composed. The angle of incidence is also known as the cone angle and 1 / 2 of this value is designated by the symbol μ. The refractive properties of a lens are summed up in a measurement known as the refractive index (n). The refractive index is a function of the bending of light from air through glass and back again.

 

3. Confocal microscopy

Confocal microscopy is an imaging technique which uses a spatial pinhole to increase micrograph contrast and/or to reconstruct three-dimensional images by eliminating out-of-focus light or flare in specimens that are thicker than the focal plane. The principle of confocal imaging was patented by Marvin Minsky in 1961. It is an integrated microscope system consisting of a fluorescence microscope, multiple laser light sources, a confocal box or scans head with optical and electronic equipment, a computer and monitor for display, and software for acquiring, processing, and analyzing images (Figure 17.3).

 

3.1. Principle

In this type of microscope, the specimen is illuminated by a finely focussed laser beam through a pin hole that rapidly scans across the specimen at a single depth, thus, illuminating only a thin plane or optical section within the specimen. Short wavelength incident light is absorbed by the specimen and remitted at longer wavelengths. Lights emitted from the specimen are brought to focus at a site within the microscope that contains a second pinhole aperture. The light emitted from the illuminated plane of the specimen passes back, up through the objective lens, through the dichroic mirror and emission filter, and through a second pin hole. Light rays that might emanate from above or below this plane are prevented from participating in image formation. As a result, out – of – focus points in the specimen becomes invisible. The emitted light is finally detected by a photomultiplier behind the second pin hole. The two pin holes – illuminating and detecting – are located in planes conjugate to the plane of focus of the image.