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Light Microscopy: Instrumentation and Principles

Author(s): David R. Caprette, PhD

Compound Light Microscope

A compound microscope employs multiple lenses that combine to produce a high quality, well resolved, magnified image. A component called a condenser collects light from an external or internal source and projects it toward a specimen. A condenser is essential because it modifies the light beam to match the properties of the objective lens. The user can control and optimize contrast and resolution by adjusting the condenser. If specialized optics are available, their use requires changing a condenser position or exchanging condensers.

Light from a specimen passes into an objective lens that magnifies the image. A good quality objective lens (often simply called an objective) is composed of multiple individual elements, producing a much better resolved and corrected image than one could obtain using a simple lens. The user should have a choice of lenses, arranged on a turret in order of increasing magnification. Specialized objectives that are required for phase contrast or D.I.C. microscopy usually can produce bright field images as well.

An eyepiece lens (called an ocular) magnifies the image from the back lens of the objective. Final magnification is the product of the objective magnification and ocular magnification. For example, a 20 power (20x) objective magnifies the image of a specimen twenty times. A 10x ocular magnifies the magnified image 10 times further. In this example the final magnification is 20 x 10 = 200x.

Well designed modern microscopes are equipped with a binocular eyepiece tube (i.e., two oculars) so that one can view with both eyes. One's acuity is much better using both eyes in the natural fashion than when squinting with one eye down a monocular eyepiece tube. A measuring device, called a reticule, may be placed in an ocular to aid in counting or measuring dimensions of objects.

These features are common to all types of light microscopes, including bright field, dark field, phase contrast, polarizing, and fluorescence microscopes, as well as instruments that combine two or more optical systems. The least expensive option when equipping a teaching lab is to purchase dedicated bright field microscopes with no other special features. Of all of the other options, dark field is probably the most versatile and least expensive upgrade, but it is highly underused. A combined bright field/dark field microscope permits a user to see virtually any biological specimen at the cellular or tissue level, living or dead, stained or unstained.