All to frequently, sophisticated and well-equipped microscopes fail to yield excellent images due to incorrect use of the light source, which usually leads to inadequate sample illumination. When optimized, illumination of the specimen should be bright, glare-free, and evenly dispersed in the field of view.
There are numerous light sources available to illuminate microscopes, both for routine observation and critical photomicrography. A most common light source, because of its low cost and long life, is the 50 or 100 watt tungsten halogen lamp as illustrated at the base of the microscope diagram in Figure 1, which also details the optical pathways in a typical modern transmitted light microscope. In this figure, the tungsten-halogen lamp emits a continuous spectrum of light centered at 3200 K (when set at a lamp voltage of +9 volts), which is then passed through a collector and field lens before being reflected into the substage condenser and onto the specimen. Image forming light rays are captured by the microscope objective and passed either into the eyepieces or directed by a beamsplitter into one of several camera ports. Throughout the optical pathway of the microscope, illumination is directed and focused through a series of diaphragms and lenses as it travels from the source to illuminate the specimen and then into the eyepieces or camera attachment. Alignment of the optical components of a microscope to optimize illumination in modern microscopes is carried out following the rules ofKöhler illumination. More details of how both transmitted and reflected light microscopes are aligned for properKöhler illuminationare discussed in our sections on setting up a microscope fortransmitted lightandreflected light. The optical pathways shown above in Figure 1 are typical for a transmitted light microscope and involve a number of lenses, diaphragms, mirrors, and beamsplitters to direct light through the microscope.
Tungsten-halogen lamps are relatively bright with a color spectrum centered at 3200 K (when set at approximately +9 volts), but require color conversion filters to raise their color temperature to daylight equivalence. Another popular light source is the 75 to 150 watt xenon lamp because of its very high brightness and long life, a relatively even output across the visual spectrum, and a color temperature which approximates that required by daylight film emulsions. Where very high light intensity is required, tin-halide lamps are often used. Influorescencemicroscopy, particular for the purpose of critical photomicrography, 100 watt or 200 watt mercury burners are often employed. In former years, carbon arc lights or zirconium bulbs might have been used, but these sources are seldom seen today. For more information and a detailed discussion of the wide spectrum of lamps available for microscope illumination, visit our section onlight sourcesfor microscopy and photomicrography.
Alignment of the source illumination through optical pathways in reflected light microscopy is also of primary concern, particular with respect to metallography, semiconductor wafer inspection, and the remarkable new progress being made in fluorescence microscopy. Reflected light microscopes are also illuminated with a variety of light sources (as discussed above) and can be adjusted for optimal performance using Köhler illumination. This is discussed in greater detail in our section on configuration ofKöhler illumination for reflected light microscopy. Illumination pathways for reflected light microscopy are also the topic of several interactive Java tutorials including the one linked directly below.
In his excellent book "Photography Through the Microscope",John Dellyargues that between 80 and 90 percent of all photomicrographs submitted to contests, exhibitions, and scientific publications are victims of improperly aligned optics resulting in poor sample illumination. This is one of the most common problems with microscopy and photomicrography in general, and a surprise examination of the student microscopes in many university laboratories will reveal an abundance of poorly adjusted substage condensers and illumination sources. We suspect that incorrect alignment and adjustment of both the substage condenser and field diaphragm, over the entire range of objectives in a given microscope, are the biggest source of errors in photomicrography.
The necessity for proper illumination cannot be overemphasized. All too often, a $25,000 microscope is reduced to the level of a hand magnifier by improper illumination and alignment, resulting in photomicrographs that are surpassed by those taken with a $2000 microscope under optimum conditions ofKöhler illumination. In the companion sections of this primer dealing with the theory of Köhler illumination and its implementation (both fortransmittedandreflectedlight) we discuss critical aspects of microscope configuration and have included a variety ofinteractive
