Microscope Optics

Written by microscopes

Saturday, 04 August 2007

Information on Microscope Optics.

Microscope Optics

The microscope is an optical instrument designed for the purpose of enlarging details to such an extent that they may be clearly discerned by the eye. It may be simple or compound, depending upon whether it contains one or more lenses. A simple microscope is usually found in the form of one double convex lens and is commonly called a magnifying glass. The compound microscope differs from this in that it has several lenses, each magnifying the image of the other until great enlargement is secured. It consists essentially of one lens, the objective, close to the subject, which forms an image which is in turn magnified by another lens, the ocular or eyepiece. Reference to Fig. I will help to make this clear.

In this diagram the objective O forms an image of the specimen on the slide S, at the plane PI. This image, if allowed to reach the eye, would be inverted and a real image. Before it can be formed, however, the light rays encounter the lower lens of the eyepiece B which, in com- bination with the upper lens C, produces a magnified virtual image at the plane P 2 , corresponding to the real image P^ Thus the magnifying power is the product of the separate magnifying powers of the two lens systems, or that of the objective multiplied by that of the eyepiece.

Thus it would appear that any magnification desired, however great, could be secured simply by increasing the magnifying power of the two lens systems. It would seem at first sight that there is no limit to the amount of detail we could perceive, for could we not use another micro- scope to magnify the image produced by the first and thus secure unlimited magnification? Magnification yes, but detail no, for, unfortunately, the amount of detail which may be discerned is limited by optical laws. Mere mag- nification of the subject does not enable us to see more detail. This quality, known as resolving power, is de- termined by the construction of the objective.

Fig. I. Diagram of the light path through a microscope.

An eyepiece with a magnification of ten times, that is, one which magnifies the objective image ten times, will give about all the detail that the objective is capable of resolving. This limit of resolving power is fixed by the nature of light itself. Light is not a continuous flow of substance. It consists of definite waves of definite wave- length. This gives to light, in a manner of speaking, a certain structure which makes it impossible to see things that are smaller than the structure of light itself. Re- solving power may be defined as the distance by which two small elements in an object must be separated in order to be visible, and is a function of what is known tfs the numerical aperture of the lens.

In microscopical writings the term numerical aperture is abbreviated to N.A. 'The higher the N.A. the greater the resolving power and the finer the detail which is re- vealed. Numerical aperture is equal to the effective aperture of the back lens of the objective divided by twice the equivalent focus. Thus if a very narrow pencil of light is used for illumination, the finest detail which may be revealed by a microscope of sufficient magnification is equal to ^L in which wl is the wavelength of the light used for illumination. As the pencil of light becomes wider, the resolving power is increased until a maximum is reached when the whole aperture is filled with light. In this case the resolving power is twice as great, as repre- sented by the formula 2 -^ ] A - This same limit is reached when a narrow pencil of light enters the lens as obliquely as possible. The wavelength of light may be taken as one half of i / 1,000 of a millimeter, or about i 750,000 of an inch. If then we assume a lens in which the effective aperture of the back lens is equal to the equivalent focus, the lens will have an N.A. of 0.5. This lens can separate lines which are 1/25,000 of an inch apart if the back lens is filled with light, but if a narrow pencil is used the lines must be only 1/12,500 of an inch apart to be resolved by this objective. So we see that extremely high resolving power requires objectives of wide numerical aperture, in the order of i.o N.A., which will resolve 50,000 lines to the inch. Use of such objectives calls for special equip- ment and manipulation.

In using a microscope we look through a sheet of glass, the cover glass over the specimen. While this is trans- parent it may act also as a reflector if the light passing through it strikes it at an angle greater than a certain fixed angle. For the same reason, and more readily be- cause of the black background provided by the inside of the microscope tube, the lens of the objective may become a reflector. In order to control this angle and provide a definite path for the light to travel we equip the micro- scope with a condenser (the substage condenser) placed in the path of the light. Thus we may control the light and regulate the amount so that it just fills the rear ele- ment of the objective when it is examined with the eye- piece removed from the tube. We also place a drop of oil on the cover glass and immerse the objective in this. The oil, having the same refractive index as the glass, presents a homogeneous material through which the light may travel in its own medium, thereby preventing the dis- persion which would otherwise take place. Such lenses are known as oil-immersion lenses and by their use nu- merical apertures as high as 1.4 may be attained, which, under the most favorable conditions, permit resolutions of the order of 100,000 lines to the inch. These objectives are available only on the most expensive professional microscopes, so the beginner need not search for them as accessory equipment to amateur instruments. This di- gression into numerical aperture is included solely for the fortunate possessors of more pretentious micro- scopes in order to clear up the terms used in catalog de- scriptions of such equipment.