Laser Scanning Confocal Microscopy

Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. Modern instruments are equipped with 3-5 laser systems controlled by high-speed acousto-optic tunable filters (AOTFs), which allow very precise regulation of wavelength and excitation intensity. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible and near-infrared spectral regions, these microscopes are capable of examining fluorescence emission ranging from 400 to 750 nanometers. Instruments equipped with spectral imaging detection systems further refine the technique by enabling the examination and resolution of fluorophores with overlapping spectra as well as providing the ability to compensate for autofluorescence. Recent advances in fluorophore design have led to improved synthetic and naturally occurring molecular probes, including fluorescent proteins and quantum dots, which exhibit a high level of photostability and target specificity. Introduction and Historical Perspective The technique of laser scanning and spinning disk confocal fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available using other contrast modes with traditional optical microscopy (1-12). The application of a wide array of new synthetic and naturally occurring fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity amid non-fluorescing material (13). In fact, the confocal microscope is often capable of revealing the presence of a single molecule (14). Through the use of multiply-labeled specimens, different probes can simultaneously identify several target molecules simultaneously, both in fixed specimens and living cells and tissues (15). Although both conventional and confocal microscopes cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved. The basic concept of confocal microscopy was originally developed by Marvin Minsky in the mid-1950s (patented in 1961) when he was a postdoctoral student at Harvard University (16, 17). Minsky wanted to image neural networks in unstained preparations of brain tissue and was driven by the desire to image biological events at they occur in living systems. Minsky’s invention remained largely unnoticed, due most probably to the lack of intense light sources necessary for imaging and the computer horsepower required to handle large amounts of data. Following Minsky’s work, M. David Egger and Mojmir Petran (18) fabricated a multiple-beam confocal microscope in the late 1960s that utilized a spinning (Nipkow) disk for examining unstained brain sections and ganglion cells. Continuing in this arena, Egger went on to develop the first mechanically scanned confocal laser microscope, and published the first recognizable images of cells in 1973 (19). During the late 1970s and the 1980s, advances in computer and laser technology, coupled to new algorithms for digital manipulation of images, led to a growing interest in confocal microscopy (20). Fortuitously, shortly after Minsky’s patent had expired, practical laser scanning confocal microscope designs were translated into working instruments by several investigators. Dutch physicist G. Fred Brakenhoff developed a scanning confocal microscope in 1979 (21), while almost simultaneously, Colin Sheppard contributed to the technique with a theory of image formation (22). Tony Wilson, Brad Amos, and John White nurtured the concept and later (during the late 1980s) demonstrated the utility of confocal imaging in the examination of fluorescent biological specimens (20, 23). The first commercial instruments appeared in 1987. During the 1990s, advances in optics and electronics afforded more stable and powerful lasers, high-efficiency scanning mirror units, high-throughput fiber optics, better thin film dielectric coatings, and detectors having reduced noise characteristics (1). In addition, fluorochromes that were more carefully matched to laser excitation lines were beginning to be synthesized (13). Coupled to the rapidly advancing computer processing speeds, enhanced displays, and large-volume storage technology emerging in the late 1990s, the stage was set for a virtual explosion in the number of applications that could be targeted with laser scanning confocal microscopy. Modern confocal microscopes can be considered as completely integrated electronic systems where the optical microscope plays a central role in a configuration that consists of one or more electronic detectors, a computer (for image display, processing, output, and storage), and several laser systems combined with wavelength selection devices and a beam scanning assembly. In most cases, integration between the various components is so thorough that the entire confocal microscope is often collectively referred to as a digital or video imaging system capable of producing electronic images (24). These microscopes are now being employed for routine investigations on molecules, cells, and living tissues that were not possible just a few years ago (15). Confocal microscopy offers several advantages over conventional widefield optical microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick specimens. The basic key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light or glare in specimens whose thickness exceeds the immediate plane of focus. There has been a tremendous Claxton, Fellers, and Davidson CONFOCAL MICROSCOPY Figure 1. Comparison of widefield (upper row) and laser scanning confocal fluorescence microscopy images (lower row). Note the significant amount of signal in the widefield images from fluorescent structures located outside of the focal plane. (a) and (b) Mouse brain hippocampus thick section treated with primary antibodies to glial fibrillary acidic protein (GFAP; red), neurofilaments H (green), and counterstained with Hoechst 33342 (blue) to highlight nuclei. (c) and (d) Thick section of rat smooth muscle stained with phalloidin conjugated to Alexa Fluor 568 (targeting actin; red), wheat germ agglutinin conjugated to Oregon Green 488 (glycoproteins; green), and counterstained with DRAQ5 (nuclei; blue). (e) and (f) Sunflower pollen grain tetrad autofluorescence.