Contrast Enhancement in Light Microscopy

Optical microscopes, which are among the oldest instruments of scientific discovery, continue to be key tools in both biomedical research and routine diagnosis. This remains true despite the development of a wide range of new imaging technologies, many with far greater resolution—ranging from electron microscopes to the multitude of scanning probe systems available today. The overall simplicity of optical microscopes, their minimally destructive impact, particularly on live specimens, and the many microscopy techniques available for enhancing or visualizing specific specimen features make this technique exceptionally useful in biomedical applications. In recent years, electronic imaging has both greatly enhanced the capabilities of light microscopes and placed ever-increasing demands on their optical performance. A number of purely optical methods can be used to enhance feature extraction from biological material for both visual observation and subsequent electronic image processing. In the present discussion, a short primer on image formation in basic transmitted-light bright-field microscopes and their resolution limits will be followed by a brief outline of how to optimize the imaging conditions by using what is known as Köhler illumination, and finally by discussion of alternatives to bright-field microscopy.