LS1151a- microscopy tutorial- general concepts- intro to light microscopes

A light microscope uses visible light to produce magnified images of cells, tissues and small organisms.

A variety of light microscopes are used in biological sciences, these include brightfield, darkfield and phase contrast microscopes.

Modern microscopes are all compound microscopes. Compound means that the magnified image of an object produced by the objective lens (nearest the object) is secondarily magnified by one or more additional lenses.

In most teaching brightfield microscopes, the objective lens forms the primary magnified image within the microscope and the ocular lens (eyepiece lens) further magnifies the primary image.

 because the two stages of magnification by the objective and ocular lenses are achieved over a short optical distance.

The curvature of biconvex microscope lenses produces a large angle of light.

The strength of the lens depends on its curvature, the > the curvature, the stronger the magnification.

The ~ total working magnification is calculated by : objective lens magnification value of X ocular lens magnification value

worked example:

Resolution of fine detail is as essential as magnification of an image by a microscope.

Resolution [of microscope] definition: shortest distance between 2 points on a specimen that can be distinguished as separate objects, so refers to the amount of detail that can be seen in an image.

(aka minimum resolved distance) is the greatest potential of an optical system to distinguish separate points.

The resolution of a microscope objective lens is governed by its numerical aperture (N.A.) and wavelength of light used (λ).

It is not possible to obtain an absolutely sharp image because diffraction of light in the specimen is always a limiting factor.

The diffraction of light is the scattering of light when light encounters an obstacle in a specimen.

The magnification and (N.A.) values are engraved on the side of each lens.

The value of the N.A of a lens is influenced by the angle of the cone of light entering the lens.

E.g. a cone of light enters a 10 x objective lens at a narrow angle. This means the light does not spread out sufficiently after leaving the microscope slide to separate out images of closely arranged objects in a specimen. This results in a low resolution of the image.

With a 40 x objective lens, the cone of light enters the lens at a wider angle after leaving the slide. This means that closely arranged objects within a specimen appear separated, resulting in a higher resolution than with the 10 x objective lens (see diagram below).

The > the value of the numerical aperture (angle of light ), the > the resolving power of a lens.

The resolution of light microscopy is limited by the radiation source used, i.e. visible light.

The best resolution of a compound microscope is 200 nm.

Electron microscopes must be used for resolutions better than 200 nm.

They use a beam of electrons which have shorter wavelengths. The < the wavelength, the better the resolution.

               the ~ best resolution of a brightfield microscope working out

(r = resolution, 0.61 = constant, λ = wavelength of light)

r = 0.2 µm or 200 nm