Understanding Conjugate Planes and Köhler Illumination

The development of advanced light microscopy techniques in recent years has been quite impressive. Cellular biologists are now able to visualize processes such as protein development in real time due to advanced techniques such as Fluorescence Recovery After Photobleaching (FRAP); they are no longer limited to indirect biochemical tests to determine sequences of biological events. However, despite the increasing sophistication of microscopy techniques, the fundamentals of microscope alignment are often neglected and misunderstood although they are vital to obtaining the best microscopy results. Correct alignment of microscope components ensures proper specimen illumination in order to achieve the best balance between image contrast and resolution. In 1893, August Köhler developed a technique still used today for aligning microscope components that results in optimal specimen illumination. It is important to understand more than the procedural steps of Köhler Illumination; being familiar with the concepts behind these steps will foster the knowledge necessary to effectively improve image quality for challenging specimens. An understanding of the illumination and image-forming pathways is necessary in order to realize the full imaging potential of microscopes. In a properly focused and aligned optical microscope, a review of the geometrical properties of the optical train demonstrates that there are two sets of principal conjugate focal planes that occur along the optical pathway through the microscope. One set consists of four field planes and is referred to as the field or image-forming conjugate set, while the other consists of four aperture planes and is referred to as the illumination conjugate set. Each plane within a set is said to be conjugate with the others in that set because they are simultaneously in focus and can be viewed superimposed upon one another when observing specimens through the microscope. Presented in Figure 1 is a cutaway diagram of a modern microscope (a Nikon Eclipse E600), which illustrates the strategic location of optical components comprising Introduction National High Magnetic Field Laboratory, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32306, [email protected], http://microscopy.fsu.edu