Understanding Immersion Oil
Microscopes use lenses to magnify small objects. At high magnifications, light scatters as it passes from the glass slide, through the air, and into the objective lens. This scattering reduces light collected, leading to a blurry image and loss of fine detail. To overcome this, oil immersion involves placing a special oil between the specimen slide and the objective lens. This method significantly improves image resolution, allowing observation of minute structures like bacteria or cellular organelles.
Immersion oil works by matching the refractive index of glass, typically around 1.52. Light bends when traveling between mediums with different refractive indexes. Air (refractive index 1.00) causes significant bending when light moves from the glass slide (1.52) to air and then into the glass lens (1.52). By introducing immersion oil, with a refractive index close to glass, the light path becomes more uniform. This minimizes light bending and scattering, ensuring more light rays enter the objective lens for a clearer, higher-resolution image.
Various types of immersion oils exist, each with specific properties. Synthetic oils are often preferred as they are non-drying and non-fluorescing, suitable for a broader range of microscopy techniques. Always use the correct oil type specified by the microscope and lens manufacturer. Using an incompatible oil can damage the objective lens or distort image quality over time.
Microscope and Slide Preparation
Proper preparation of the microscope and specimen slide is essential for optimal viewing and to prevent damage. Begin by placing your prepared specimen slide onto the microscope stage, securing it with the stage clips. This ensures the slide remains stable throughout observation.
Next, locate the specific area of interest on your slide using lower magnification objectives. Start with the lowest power objective (typically 4x or 10x) and use the coarse focus knob to bring the specimen into general focus. Once clear, gradually move to a higher power objective, such as 40x, refining the focus with the fine focus knob.
After identifying and focusing on your desired region at 40x magnification, rotate the revolving nosepiece. Position it so the 40x objective moves slightly past the viewing position, creating an open space above your area of interest. This provides ample room to safely apply immersion oil without contaminating lower power objectives or risking slide damage.
Applying and Focusing with Oil
With the microscope and slide prepared, precisely apply immersion oil and focus with the high-magnification objective. Place a single, small drop of the appropriate immersion oil directly onto the cover slip, over the specimen area. Using more than one drop is unnecessary and can lead to excessive oil spread, making cleanup more difficult.
Once the oil is in place, slowly rotate the revolving nosepiece until the 100x oil immersion objective lens dips into the oil drop. This movement should be smooth and controlled to avoid trapping air bubbles, which can significantly obscure your view. The lens will make contact with the oil, creating a continuous optical path.
After the 100x objective is immersed, adjust the fine focus knob to bring the image into sharp clarity. At this high magnification, only the fine focus knob should be used, as the coarse focus knob can damage the lens and specimen. Minute adjustments of the fine focus will reveal intricate details. For optimal illumination and contrast, adjust the condenser aperture diaphragm and light intensity to enhance image quality.
Cleaning and Storage
Proper cleaning of the oil immersion lens and specimen slide after use is important for maintaining microscope longevity and performance. Immediately after observations, rotate the 100x objective away from the oil drop. This prevents oil from drying on the lens, which can harden and become difficult to remove later.
To clean the objective lens, use specialized, soft, lint-free lens paper and a designated lens cleaning solution. Apply a small amount of solution to lens paper, then wipe the objective lens in a circular motion, moving from the center outwards. Avoid harsh solvents or tissues not designed for optics, as these can scratch the lens or dissolve adhesives.
The specimen slide should also be cleaned thoroughly to remove all traces of immersion oil. Use a soft, lint-free cloth or lens paper moistened with a suitable cleaning solution, such as xylene or a specific slide cleaner, to wipe oil from the cover slip and slide surface. After cleaning, store the microscope in a dust-free environment and ensure the immersion oil bottle is tightly capped to prevent evaporation or contamination.
References
1. Refractive Index of Immersion Oils. Cargille Laboratories.
2. Microscope Care and Maintenance. Olympus.