Confocal microscopes have evolved over the past 25 years from the early stage scanning systems to a collection of sophisticated laser scanning systems designed for a range of biomedical applications. Major improvements to the photon efficiency of the instrumentation coupled with the development of novel fluorescent reporters have enabled multidimensional imaging of living cells and tissues.

Looking Back

When BioTechniques made its debut in 1983, confocal microscopy had yet to become established in the world of biological imaging. Immunofluorescence microscopy was popular, and images of the distribution of fluorescent labels attached to antibodies to specific proteins were filling the journals with spectacular grayscale images (1). The distribution patterns of many different proteins, especially cytoskeletal proteins, were giving novel views and insight into cellular structure and function at the level of the light microscope (2). Around this time, fluorescent dyes were being developed that were sensitive to cellular physiology, such as calcium ion concentration (3).

But there was a problem. The clearest images were collected from the thinnest regions of flattened cells growing on a glass coverslip in culture. The technique suffered from resolution issues when imaging even the thicker regions around the nucleus of cultured cells ((Figure 1)A). This was because the fluorescent signal from regions above and below the focal plain contributed to the images, and at best, blurred them, and often swamped out the signal completely. Most images of thicker specimens such as dividing cells, eggs, and embryos appeared as a bright fluorescent glow with little or no visible structure at all. Often structures of interest were visible in the light microscope but it was impossible to record them for publication. Researchers were forced to compromise by either physically cutting sections of their specimens or artificially flattening them in order to resolve structures of interest. Imaging living cells for structural and physiological studies was fraught with difficulty.

Figure 1.

There was a desperate need to improve the resolution of images collected using fluorescence microscopy. One possibility was confocal microscopy. The technique had been around for many years before 1983: in 1957 Marvin Minsky had patented an instrument designed for studying neural networks in the living brain, but it was not sensitive enough for fluorescence and was not well-suited to biological specimens since the stage rather than the light beam moved, causing vibration artifacts (4,5). In 1983, however, the technology was not sufficiently well-advanced to produce a confocal instrument suitable for collecting images of fluorescently labeled specimens on a routine basis. But it was close.

Coming of Age

The practical application of confocal microscopy for biological fluorescence imaging was introduced some four years later in 1987 with the introduction of the first commercially available laser scanning confocal microscopes (6,7,8). The approach was typified by the MRC 500 laser scanning confocal microscope (Bio-Rad Microsciences, Hemel Hempstead, England), which was designed to interface with an existing epifluorescence microscope rather like a video camera. The instrument consisted of a scanning unit that contained the electronics to drive two mirrors to produce a focused scanning beam in the specimen from an air-cooled argon laser together with photomultiplier tube detectors (PMT). The output from the PMTs was built into an image using a framestore card in a microcomputer, and the resulting digital image was displayed on the screen of the computer at a rate of about one full 768 × 512 pixel frame per second. Hard copies of the image files were collected by photographing the screen of the video monitor and archived digitally.

The images obtained using the instrument were a strong demonstration of the power of the confocal approach for fluorescently labeled specimens. Optical sections that showed subcellular details from within thick and brightly labeled specimens were obtained for the first time from a range of fixed cells and tissues, and with little additional specimen preparation as compared with that required for conventional epifluorescence microscopy. The liberal application of antibleaching agents, however, was highly recommended.