Immersion oil is a specialized, transparent liquid used in light microscopy to enhance the clarity and resolution of magnified images. It creates a continuous optical path between the microscope’s objective lens and the specimen on the glass slide. This is important when observing samples at high magnifications, where light behavior can otherwise limit image quality.
The Magnification Challenge
When light travels from one medium to another, such as from a glass microscope slide to the air, it refracts. This occurs because light changes speed as it passes through substances with different optical densities, known as refractive indices. Air has a refractive index of approximately 1.0, while glass slides and coverslips have a refractive index closer to 1.5. At lower magnifications, this refraction is not significantly problematic.
However, at high magnifications, especially 100x and above, the objective lens is very close to the specimen, and the light rays emerging from the sample spread out significantly. Many light rays, particularly those entering the air gap between the slide and the lens at oblique angles, refract away from the objective lens and are lost. This light loss results in a blurry, less detailed image and reduces the microscope’s ability to distinguish closely spaced objects (resolving power).
How Immersion Oil Works
Immersion oil addresses the magnification challenge by minimizing light refraction and scattering. It achieves this because its refractive index is nearly identical to that of glass. When a drop of immersion oil fills the space between the microscope slide and the objective lens, light passes from the glass, through the oil, and into the lens with minimal bending. This creates a uniform optical environment, allowing more light rays to enter the lens.
By reducing light loss, immersion oil significantly increases the objective lens’s numerical aperture (NA). Numerical aperture measures a lens’s ability to gather light and resolve fine details; a higher NA improves resolution and image clarity. Immersion oil enhances the microscope’s ability to discern minute structures that would otherwise appear as a blur. This optical continuity is essential for sharp, detailed high-magnification views.
Using Immersion Oil Effectively
Immersion oil is required for objective lenses with magnifications of 100x or higher, which are specifically designed for this purpose. To use it, first focus the specimen using a lower magnification objective, such as 40x. Then, rotate the objective turret so that the 40x lens is partially out of the way, creating space to apply a single drop of immersion oil onto the coverslip over the area of interest.
Carefully rotate the 100x objective into the oil, ensuring contact without trapping air bubbles. Use only the fine focus knob for minor adjustments once immersed, as the working distance is very small. After observation, clean the objective lens and slide immediately, as dried oil is difficult to remove and can damage the lens. Use specialized lens paper, moistened with alcohol if needed, to gently wipe the lens from the center outwards in a spiral motion until all oil residue is gone.