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Light Microscopy: Instrumentation and Principles

Author(s): David R. Caprette, PhD


Resolution (d) is the degree to which a microscope can distinguish fine details. The number represents the minimum distance that must separate two points for them to be distinguished by the human eye. The lenses on a compound light microscope are composed of multiple elements placed close together. A very good objective lens may contain as many as eight to ten individual lenses, while an ocular may contain two or three. The result is a single very well corrected lens that can magnify many times without blurring or distorting an image.

The resolving power of an objective lens should approach the theoretical limit of about 0.2 µm. A good 40x objective lens not only magnifies an image 40x, but also provides a resolution such that when the image is further magnified 10x by an ocular lens, the eye can distinguish objects that are separated by as little as two or three micrometers. Above 40x, an objective lens produces empty magnification without the use of immersion oil, a method that will be described later. Resolving power using a 100x oil immersion lens to achieve 1,000x final magnification can be brought very close to the theoretical limit. At resolution 0.2 µm, one can distinguish larger organelles, such as chloroplasts, mitochondria, and cell nuclei. Raising magnification beyond 1,000x using a visible light microscope produces only empty magnification.

Good quality objective lenses are expensive, ranging in price from hundreds to thousands of dollars. Quality and cost typically increase with magnification. Low magnification lenses, often called “scanning” lenses, are the least expensive and typically magnify from 3.5x to 4x. Typical lenses of intermediate magnification are 10x or 20x. “High dry” lenses are high magnification objectives that are used without immersion oil. The most common magnification is 40x, although you might find objectives, especially older ones, ranging from 35x to 40x. Oil immersion lenses typically magnify from 95x to 100x.

To preserve their function, objective lenses must be handled with extreme care. Organic solvents can dissolve adhesives or remove a lens coating. Distilled water or dilute acetic acid (e.g., vinegar) can be used along with good quality lens tissue to clean a dirty lens. It also is safe to use a cotton-tipped applicator stick, provided the tip is 100% natural cotton. The “rules” for cleaning lenses apply to any optical surface, including ocular and condenser lenses, filters, and even eyeglasses.