Increase Resolution with Immersion Oil

Microscope objectives are intended to image specimens either through air or a medium of higher refractive index between the front lens and the specimen. A gain in resolution by a factor of about 1.5 is attained when immersion oil is substituted for air as the imaging medium.

Immersion oil contributes to two characteristics of the image viewed through the microscope: finer resolution and brightness. Immersion oil is intended for use with "oil" objectives and the objective will have "oil" printed on it. Water immersion objectives are typically marked with a "W" and those must be used with water matching liquid. "Dry" should not be used with any immersion oils.

The advantages of oil immersion objectives are severely compromised if the wrong immersion fluid is utilized. Microscope manufacturers produce objectives with tight tolerances to refractive index and dispersion, which require matching values in the liquid placed between the cover glass and objective front lens. It is advisable to employ only the oil intended by the objective manufacturer, and to not mix immersion oils between manufacturers to avoid unpleasant artifacts such as crystallization or phase separation.

Microscope Immersion Oil Options

Which Objectives work with Immersion Oil?

The microscope objective will be engraved OIL, OEL, or HI if the objective is designed to function with immersion oil. If not, the objective is meant to be used dry. Remember to clean the immersion oil objective after use.

Help Selecting the Correct Immersion Oil

Standard Light Microscopy - Immersion Oil Type A and Type B are recommended for standard light microscopy applications.  What exactly is the difference between Immersion Oil Type A and B?

• Immersion Oil Type A, at 150 centistokes (cSt), reduces any tendancy to trap air, which is important because air bubbles can cause image degradation. Immersion Oil Type A is helpful for beginning students.
• Immersion Oil Type B, at 1250 centistokes (cSt), is thick enough for viewing multiple slides with one application, which saves time in batch processing.

Automated Hematology Systems - Immersion Oil Type 300 is manufactured to meet the stringent requirements of Automated Hematology Systems including specialized viscosity and exacting controls for consistency. Automated hematology systems require accurate, precisely controlled physical and optical properties of immersion oil for successful imaging and mechanical processing.

Inverted, Inclined, Projection, and Long Focus Instruments - Immersion Oil Type NVH and Immersion Oil Type OVH have very high viscosities (Type NVH 21,000 cSt and Type OVH 46,000 cSt), which become more desirable as the gap between the cover glass and objective increases, or between the slide and condenser.

Fluorescence Microscopy - Extremely low fluorescence is achieve with Immersion Oil Type LDF and Immersion Oil Type HF. Type HF (700cSt) is slightly more fluorescent than Type LDF (500 cSt), but is halogen free.

Elevated Temperatures (>23ºC to 37ºC) - Immersion Oil Type 37 is ideal for elevated temperatures from substage illuminators or hot stages. Developed specifically for working at human body temperatures, Type 37 has a refractive index of 1.515 and a viscositiy of 1,250 cSt at 37ºC, solving the problem of image degradation above the standard calibration temperature of 23ºC.

Oil Immersion and Refractive Index

One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. Most objectives in the magnification range between 60x - 100x are designed for use with immersion oil. Good results have been obtained with an oil that has a refractive index of n = 1.518, which is close to the refractive index of glass. All reflections on the path from the object to the objective are eliminated in this way. If this trick were not used, reflection would always cause a loss of light in the cover slip or on the front lens in the case of large angles.

Numerical Aperture (abbreviated NA) provides a convenient indicator of the resolution for any particular objective. Numerical aperture is generally the most important design criteria (other than magnification) to consider when selecting a microscope objective. Values range from 0.025 for very low magnification objectives (1x - 4x) to as much as 1.6 for high-performance objectives utilizing specialized immersion oils. As numerical aperture values increase for a series of objectives of the same magnification this results in a greater light-gathering ability and increase in resolution.

Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated. Typical immersion oils have a refractive index of 1.51 and a dispersion similar to that of glass coverslips. Therefore, light rays passing through the specimen encounter a homogeneous medium between the coverslip and immersion oil and are not refracted as they enter the lens, but only as they leave its upper surface.

Properly designed oil immersion objective lenses also correct for chromatic defects that are introduced by the first two lens elements, while introducing a minimum amount of spherical aberration. The fact that the light cone is partially converged before entering the first lens element aids in the control of spherical aberration. It should be noted that employing an oil immersion objective without the application oil between the coverslip and first lens element results in defective images. This is due to refraction that occurs at the surface of the front lens, which introduces spherical aberration that cannot be corrected by subsequent lens components within the objective. 

References: Rudi Rottenfusser - Zeiss Microscopy Consultant. Tadja Dragoo, Erin Wilson, and Michael W. Davidson - National High Magnetic Field Laboratory.

If you have any questions about microscope immersion oil or which type is best for your application, please contact Microscope World.

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