Oil immersion microscopy is a specialized technique used in light microscopy to achieve maximum clarity and detail when viewing extremely small specimens. This method increases the resolving power of the instrument, which is necessary for observing fine cellular structures, such as bacteria, that are too small to be seen distinctly with lower power objectives. The technique works by creating an uninterrupted path for light rays traveling from the sample to the objective lens.
The Typical Magnification Level
The technique of oil immersion is paired with the highest power objective lens found on a standard compound microscope, which is marked 100x. Total magnification is determined by multiplying the objective lens magnification by the eyepiece magnification, typically 10x. Therefore, the total practical magnification achieved using the oil immersion objective is 1000x. This level is the limit of useful magnification for a conventional light microscope.
The Physics of Immersion Oil
The need for immersion oil stems from the physics of light as it passes through different media, specifically the glass slide and the air gap above it. When light rays pass from a medium with one refractive index, such as glass, into a medium with a different refractive index, like air, the rays bend or scatter away from the objective lens. This bending, known as refraction, causes a significant loss of light and a corresponding loss of image resolution at high magnifications.
Immersion oil is a transparent liquid formulated to have a refractive index of approximately 1.51, which closely matches the refractive index of the glass slide and coverslip. By placing a drop of this oil between the specimen and the objective lens, the air gap is replaced, and the light travels through a homogeneous optical pathway from the slide, through the oil, and into the lens. This minimizes refraction, allowing more light rays to be captured by the objective lens.
The increase in captured light rays is quantified by the Numerical Aperture (NA) of the objective, which measures its light-gathering ability. The NA is calculated based on the refractive index of the medium. Since the oil’s refractive index (1.51) is significantly higher than that of air (1.0), using immersion oil dramatically increases the objective’s NA. A higher NA translates directly to better resolving power, enabling the clear visualization of structures separated by distances as small as 0.2 micrometers.
Essential Steps for Use
The process begins by locating and focusing on the area of interest using a lower power objective, such as the 10x or 40x lens. The field of view must be centered precisely before moving to the highest magnification. After the specimen is in focus at 400x total magnification, the nosepiece is rotated to move the 40x objective out of the way.
A single, small drop of the specialized immersion oil is placed directly onto the coverslip, precisely over the centered area of the specimen. The 100x objective is then rotated carefully into position, allowing its tip to contact and submerge into the oil drop. Once the 100x objective is immersed, the user looks through the eyepieces and uses only the fine focus knob to achieve a sharp image.
The objective lens is designed to be parfocal, meaning that the image should remain relatively close to focus when switching between objectives, requiring only minor adjustments. It is important to avoid using the coarse focus knob once the oil objective is engaged, as this can easily drive the lens into the slide and damage the glass or the objective itself.
Consequences of Improper Use
The most immediate consequence of misuse is a blurry image and a loss of resolution if the oil is not used with the 100x lens. Mixing different types of immersion oils, or using contaminated oil, can introduce air bubbles or change the optical properties. This results in image artifacts and poor contrast.
A more serious consequence is the contamination of lower-power objectives, such as the 40x lens, which are not designed to be used with oil. If the 40x objective is accidentally rotated into the oil drop on the slide, the oil can coat its front element. Since these “dry” objectives are not sealed like the oil immersion lens, the oil can seep into the lens barrel. Immersion oil can dissolve the specialized cement used to hold the internal lens elements together, leading to irreversible damage that requires costly repair or replacement of the objective.
Meticulous cleaning of the 100x objective is necessary immediately after use. The oil must be removed correctly using lens paper, sometimes followed by a small amount of lens cleaning solution to prevent dried residue from accumulating. Failure to remove the oil allows it to migrate to other microscope parts, compromising the performance and longevity of the instrument.