Confocal microscopy: comparisons, applications, and problems.

INTRODUCTION Over the past several years, fluorescence microscopy has become a primary quantitative tool in the armamentarium of the biomedical research scientist. In the past, the technology was limited to easily quenched fluorophores such as fluorescein and rhodamine, non-optimized objectives, filters consisting of little more than colored glass, and film as a recording media. At the present time, designer fluorophores span the entire visible spectrum to the near infrared, objectives are specifically designed to maximize throughput from fluorescent specimens, filters are made from specifically designed dielectric-coated glass, and modern cameras allow detection of vanishingly low numbers of photons such that a high-quality image may be recorded in a fraction of a second. Improvements in confocal microscopy have paralleled the rapid advances in wide-field fluorescence microscopy. The goal of this review is a brief history and comparison of confocal technologies, as well as a few examples of the primary applications of confocal approaches. Whereas confocal microscopes were initially only able to image one or two colors at relatively low speed, the modern confocal microscope is capable of collecting high-quality multispectral images across the visible range. The image shown in Figure 1 illustrates the high-power, high-resolution, and multicolor capabilities of the modern confocal microscope. Furthermore, specialized instruments work at very high speed and are thereby able to image living specimens with little photodamage. In this review we discuss the basic technology inherent to all confocal microscopes, the applications in which the confocal is an essential tool, with particular attention to the specialized use of confocal systems with in vivo preparations, and provide a limited comparison of the applications of specific subtypes of confocal microscopes.