When observing small structures under a microscope, the ability to distinguish between two closely spaced points is limited by resolution. Standard microscopes often produce blurry or indistinct images at the highest magnifications. This limitation is not due to the lens magnification itself, but to an optical problem related to how light passes from the specimen to the objective lens. To overcome this hurdle in high-power microscopy, a transparent fluid called immersion oil is used to maximize the light collected from the sample.
Understanding Microscope Resolution
Microscope resolution is the minimum distance separating two objects that can still be seen as distinct entities. Resolving power is limited by the wavelength of the light used for illumination and the objective lens characteristic known as the Numerical Aperture (NA). The NA measures the lens’s ability to gather light and is defined by the refractive index of the medium between the specimen and the lens, multiplied by the sine of the objective’s half-angle of light acceptance.
A higher NA value correlates directly with better resolution, allowing finer details to be seen. Since the wavelength of light is fixed, increasing the NA is the only way to improve the resolving power of the system. Objectives designed for use in air, which has a refractive index of approximately 1.0, are restricted to a maximum NA of around 0.95. To push resolution beyond this limit, the medium through which the light travels must be altered to increase its refractive index.
The Problem of Light Scatter
The physical limitation that degrades resolution occurs when light rays travel from the glass slide or cover slip into the air gap above the specimen. The glass used in microscopy has a refractive index of about 1.5. When light rays pass from this denser medium (glass) to the less dense medium (air), they bend, a phenomenon known as refraction.
This bending is particularly severe for light rays that exit the specimen at high angles. These high-angle rays carry the most detailed information about the fine structures in the specimen. Without intervention, these rays refract so sharply at the glass-air interface that they completely miss the narrow front lens of the high-magnification objective.
The loss of these high-angle rays prevents the objective lens from collecting the maximum amount of light necessary to form a detailed image. This failure to capture the full cone of light reduces the effective Numerical Aperture. The resulting image appears fuzzy and lacks fine structure, posing a significant obstacle when observing structures smaller than roughly 0.5 micrometers, which is common in microbiology and histology.
How Immersion Oil Solves the Problem
Immersion oil is a transparent fluid engineered to eliminate the light-scattering interface between the cover slip and the objective lens. The oil is formulated to have a refractive index of approximately 1.515, nearly identical to that of the glass components. Placing a drop of oil in the gap creates a continuous, homogeneous optical path from the specimen, through the glass, and into the objective lens.
Since light travels through a medium with a consistent refractive index, high-angle light rays are no longer subjected to the severe refraction that occurs at a glass-to-air boundary. These rays are directed straight into the objective lens, allowing the lens to collect a much wider cone of light. Immersion oil improves resolution primarily by maximizing the objective’s Numerical Aperture.
This technique allows objective lenses to achieve NA values greater than 1.0, with some models reaching up to 1.6. The increase in NA effectively increases the resolving power of the microscope, typically yielding an improvement in resolution by a factor of about 1.5 compared to using air. This maximization of light collection results in a brighter, clearer, and more highly resolved image, making it possible to distinguish minute details.
Practical Considerations for Use
Immersion oil is reserved for the highest-power objective lenses, most commonly the 100x objective, which is designed to work specifically with the oil. Only a single, small drop of the correct immersion oil is required, placed onto the cover slip before gently lowering the objective into the oil. Using the wrong oil, or using an objective designed for air immersion with oil, will introduce optical errors and distort the image.
Users must select the proper oil type, as different formulations exist for various applications. Type A and Type B are common for general light microscopy. Type A has a lower viscosity, reducing the chance of trapping air bubbles, while the thicker Type B is useful for viewing multiple slides sequentially.
The most important practical step after use is immediately cleaning the objective lens with specialized lens paper and an appropriate solvent, such as anhydrous alcohol. If left to dry, the oil can harden, potentially trapping debris or damaging the delicate lens coatings. Proper maintenance is necessary to preserve the objective’s optical quality.