The theoretical resolution of the light microscope was first defined by Abbe in the following equation.

Abbe's equation for theoretical resolution of the light microscope:

The actual resolution achievable with a light microscope is not as great. We will discuss the reasons for this later.

It is important to understand and to recognize the various components of the light microscope. The first and perhaps the most important element are the lenses.

Figure 2
The six simple lenses. a,b, & c are converging or positive lenses. d,e, & f are concave or negative lenses.

Figure 3
Converging and diverging lenses. Parallel rays enter a convex lens converge and meet at the principle focus. The distance between the center of the lens and the principal focus is the focal length. Parallel rays entering a concave lens diverge and never meet.

The Objective Lens is the first part of the imaging system; the objective lens forms a primary, enlarged image of the object. Very fine details are distinguished with the objective lens. The eyepiece sometimes called the ocular lens, is the second lens, which forms a secondary, further enlarged image. By multiplying the magnifying power of the objective lens and the magnifying power of the ocular the final magnification is found. A Substage Condenser lens is the third optical component. It is placed on a platform beneath the object. Light is directed through the substage condenser and converges to a point at the position of the specimen. The light rays diverge as they pass through the specimen and form an inverted cone, whose base is just large enough to fill the aperture of the objective. The size of the light beam is controlled by a diaphragm beneath the condenser called the aperture diaphragm.

The light source should contain both a lens to project an image of the lamp filament called a field condenser and a diaphragm to control the size of the illuminated field called a field diaphragm.

Figure 4
Typical lamp for light microscope.

Kohler Illumination is the most common method of illumination. In Kohler illumination the image of the source is projected by the field condenser onto the substage condenser, to the top of the plane of the object. This method assures even illumination.

Modern light microscopes use several different modes of operation depending on the needs of the investigator. The most common of these being brightfield microscopy in which direct light passes through the objective aperture and illuminates the background against which the image is seen. Since the structural elements being resolved have little variance in refractive index, the image will lack contrast and the details remain invisible. Small structure detail can be revealed by changing the absorption of the object by means of staining.

Many microscopes have special lenses that allow an investigator to image using phase-contrast. This method reveals details in specimens possessing very slight differences in optical path or refractive index in the surrounding media. Phase contrast requires no staining and can be used with living tissue. Contrast is produced in this method by phase differences in the light leaving the object to amplitude differences in the image. A phase plate alters the optical path length traveled by diffracted light from that traveled by direct light.

Figure 5
Light waves in brightfield and phase contrast after passing through an object. Pathway A represents the lightwave before encountering the object. Wave B represents the wave phase after passage in brightfield (unstained mode). C compares the wave phase of an object veiwed with phase contrast.

Differential interference contrast (DIC) differs from phase contrast in that the image has a strong relief and three-dimensional appearance. It must be remembered that the impression of surface details are the results of the optics and not the specimen for most biological samples. The optics for DIC consist of a polarizer at the light source and Wollaston prisms in the condenser and above the objectives. The beam passes through the polarizer, enters the first prism where it is split in two. One beam vibration is parallel to the prism and one is perpendicular. Both beams pass through the specimen in parallel in close proximity and are recombined in the second prism.

Figure 6
Differential Interference Contrast Schematic.