As magnification increases on a microscope the correction in refractive index is a requirement for finer resolution and brightness. As the light cone passes through the different layers of glass, sample mounting media and air light tends to bend at different angles. In most cases the sequence of the light leaving the substage condenser is glass (1.515nD), air (1.00nD), glass slide (1.515nD), sample, mounting media, cover slip (1.515nD), into the objective lens which is approximately 1.515nD.
Immersion oil has a refractive index of 1.515nD and is applied to the top of the cover slip and then the tip of the oil immersion objective is then immersed into the drop of oil. This completes the "optical connection".
The resolution obtained is directly related to the angular aperture (AA), the larger the A.A. having a wider cone with more scattered (oblique) rays. Unless there is a homogenous light path, the most oblique rays are lost to internal reflection inside the glass slide or cover slip. The use of immersion oil permits full use of the resolving power of the objective lens. Immersion oil objectives increase the resolution by approximately fifty percent over dry objectives of equivalent focal length.
The resolving power of an optical system is figured by averaging the numerical aperture (N.A.) value of the objective and the working N.A. of the condenser.
The acid value of immersion oil should be very low. If not then the higher acid content can lead to eventual deterioration of the metal parts of the objective lens or worse yet the dissolving of the cements used to manufacture the lens. This is turn leads to a leakage problem as immersion oil is wicked up into the interior of the lens.
Viscosity of the oil is up to the user. Lower viscosities tend to run and spread all over the microscopes requiring more clean up. Higher viscosities are more practical and not as messy. Very high viscosities are used for instruments that are mounted on slight angles.
When applying immersion oil use only the amount required for the individual slide. Too much oil does not help with the optical performance as much as it requires more clean up. Not enough oil will not allow a good optical contact with the objective.
Which oil is the best? Our choice is Cargille Immersion Oil. It is manufactured with the best materials. Immersion oil must meet the design requirements of older microscopes and become the design criteria for new instruments.