Immersion Oil

MA700/10, Immersion OilImmersion Oil and the MicroscopeSince the microscopist's major field of interest is the application of microscopes and related equipment, the fields in which the instruments are used are, in a sense, secondary. However, many scientists, having selected a field, find the use of a microscope a necessity but secondary to their major field of interest. Therefore, the microscope is thrust upon them as an essential tool. Often, basic background necessary for proper use of the 'tool' is lacking or inadequate, having been picked up on the job so they can 'get by.'Considering the number of microscopes being used in all types of laboratories and the number of scientists and technicians using these instruments, from reports and requests we gather that they have learned to use them in what might be referred to as 'on the job' training to the 'get by' level of proficiency.This paper will attempt to broaden the understanding of the 'business area' of the microscope, between the condenser and the objective, as it is affected by the use of oil immersion objectives, and also expand on properties of immersion oils and how they can be more fully utilized.The Function Of Immersion OilImmersion Oil contributes to two characteristics of the image viewed through the microscope: finer resolution and brightness. These characteristics are most critical under high magnification; so it is only the higher power, short focus, objectives that are usually designed for oil immersion.Oil immersion objectives are generally available from 40 to 120x. These must not be confused with 'high dry' objectives or water immersion objectives that are also made in this range. Just as an 'oil' immersion objective must be used with oil to get a usable image, a 'water' immersion objective must be used with water and a 'dry' must be used dry. The use of oil on a high dry will destroy the image by negating corrections for spherical and chromatic aberration.For any given lens there is a fixed focal length. With the objective in focus there is a cone of light extending from a point on the specimen to the full diameter of the objective lens. The angle formed by this cone is the angular aperture (A.A.), shown diagrammatically in Figure 1. It may vary from 10° for low power dry (long focus), to 140° for high power oil (short focus). the greatest theoretical angular aperture is, of course, 180° with zero focal length.Below the specimen is a second, matching, cone of light, the base of the cone being the top surface of the condenser and the apex at a point on the specimen. Theoretical illumination, then, provides a straight line path for each ray from condenser to objective lens.This straight-line path is disturbed by any material of different refractive index. Ideally, Fig. 2, progressing upward from the condenser there is air (index 1.00), slide (approximately 1.515), mounting media, cover glass (approximately 1.515), air (1.00), and finally the objective. Since most condensers and objective lenses are 1.515, a homogeneous path is obtained by filling the air gaps with an immersion oil (1.515) and using a mounting media (1.515). Mounting media other than 1.515 have advantages and disadvantages but are not a part of this discussion.The resolution obtained is directly related to the angular aperture, the larger A.A. having a wider cone with more oblique rays. However, unless there is a homogeneous optical path, the most oblique Rays are lost by internal reflection within the slide or cover glass.The oil immersion objective of medium angular aperture has more resolving power than a ' dry' with larger angular aperture and the term, proposed by Ernst Abbe, 'numerical aperture,' (N .A.) must be considered. N.A. is equal to n x sin 1/2 A.A. where n is the lowest refractive index in the path. Therefore, the air gap between cover glass and objective gives a theoretical maximum N.A. of 1.00; water, 1.33; immersion oil, 1.515. Since other limitations permit only a practical N.A. of 1.40 with lens glass 1.515, the use of immersion oil permits full use of the resolving power of the objective. And, for a given angular aperture, immersion oil objectives increase the resolution by approximately 50% over dry objectives of equivalent focal length.Just as immersion oil permits utilizing the full N.A. (resolving power) of the objective, it is also a necessity for obtaining the maximum N.A. of the condenser. With a condenser N.A. 1.40 and an air gap between the condenser and the bottom of the slide, the limiting value is N.A. 1. (air); The additional light being lost by internal reflection within the condenser.Whereas an objective must be used 'dry' or 'oiled,' depending on design, a condenser will work oiled or dry, but will be limited to a working value. Full resolution of a condenser, N.A. 1.25, can be obtained by 'oiling' with water (1.33), or immersion oil (1.515). A condenser of N.A. 1.40 must have a media of 1.40 or greater, such as immersion oil 1.515, to utilize its designed N.A.The third consideration is the mounting media itself. An 'air mounted' specimen can receive a light cone of 1.0 N.A. The excess of light is lost by total reflection at the top surface of the slide. So, there is a second limiting or working N.A. value to be considered in determining the usable N.A. of the condenser.The resolving power of an optical system is computed by averaging the N.A. value of the objective and the working N.A. of the condenser. The objective, as mentioned, must be used as designed, dry, watered or oiled. The condenser working N.A. is only equal to the designed N.A. when it is oiled to the slide, and a mounting media is used having an index greater than the condenser N.A.