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Confocal Microscopy in Biomedical Research Paul J COMMENT THIS ARTICLE SEE COMMENTS ON THIS ARTICLE CONTACT AUTHOR March 1999 (Volume 40, Number 3) Confocal Microscopy in Biomedical Research Paul J. Rigby, Roy G. Goldie Biomedical Confocal Microscopy Research Centre, Department of Pharmacology, University of Western Australia, Nedlands, Western Australia, Australia Confocal microscopy has allowed a major advance in biological imaging, since it represents a rapid, cost effective means of ecamining thick tissue specimens. In most cases, this involves fluorescence imaging and it is increasingly being used as a basic tool in biomedical research. Confocal microscopy allows the collection of thin optical sections, without the need for physical sectioning of the tissue. Additionally, confocal microscopes can usually produce images with greater sensitivity, contrast and resolution than those produced with normal light microscopes. We attempt to explain how this technology might be better used as a routine research tool. Since high quality, in-focus optical sections of thick tissue preparations can be generated quickly, confocal microscopy, in combination with immunofluorescence histochemistry, can now be used to examine complex three-dimensional distributions of distinct structures within tissues such as nerves within airways. Additionally, ultraviolet confocal microscopy allows the assessment of both dynamic and static phenomena in living cells and tissues. Thus, in addition to the imaging of fluorescence associated with structural elements, confocal microscopes can be used to quantitatively evaluate the distribution and fluxes of intracellular ions like calcium. Rapid, line-scanning confocal microscopes can be used in the assessment of dynamic events. For example, the in vivo imaging of microvascular permeability in airways becomes possible for the first time. By providing examples of some of our uses for confocal microscopy, we might encourage others to explore this relatively new and important texhnology for examining events and structures in single cells, tissue samples and in intact animals. Key words: bronchi; calcium; immunofluorescence microscopy; fluorescence microscopy; microscopy, confocal; muscle, smooth; substance P; trachea Light and electron microscopy have long been important research tools used in analyzing biological specimens and both techniques have provided valuable insights into cellular biology, structure, and function. Although electron microscopy offers excellent resolution, artifacts introduced in specimen preparation (fixation and sectioning) can severely limit some applications of this technique to biological and, in particular, to living specimens. Conventional wide field microscopy is routinely used to examine living tissue, but suffers from the physical resolution limits imposed by using visible light as the illumination source. Additionally, conventional microscopy of living or thick specimens suffers enormously from out-of-focus light degrading the image quality. Furthermore, in the absence of enormous operator effort, both of these techniques reveal little information about the three dimensional nature of biological specimens. In 1957, the first confocal microscope was patented by Marvin Minsky, and commercial systems for cell biologists only became available in 1987 (1). These developments heralded a quantum leap in the quality of light microscopic data that could be obtained from biological specimens. The impact of the enormous improvement in the efficiency of data collection and in the quality and power of the data provided to the researcher by such efficient image production and processing, cannot be over- estimated. It is perhaps little wonder that confocal microscopy has become so popular in less than ten years. Confocal Microscopy: Principles and Advantages Traditionally, in conventional microscopy, a condensor lens is used to uniformly and simultaneously illuminate a wide area and volume of the specimen (hence the term wide-field microscopy). In thick specimens, this results in out-of-focus blur originating from volumes above and below the plane of focus. This out-of-focus light can reduce contrast and resolution and can severely detract from the interpretation of fine microscopic detail. In an effort to reduce these problems, conventional microscopy has routinely utilized thin or ultra-thin sections of tissues to enable clear views of the tissue structure to be obtained. In confocal microscopes, the illumination is both sequential and focused on a small volume of the tissue, with regions away from the focal plane receiving less illumination, thus reducing the out-of- focus blur. Additionally, both the illumination and detection systems are focused on the same single volume element of the specimen (2). Thus, the illumination, specimen, and detector are all focused on the same volume and are therefore confocal. With the addition of a carefully aligned aperture at a focal point in the optical path, further reduction in out-of-focus information is achieved (Fig 1). Figure 1. The principle of confocal microscopy. A light source, typically a laser, is reflected by a dichroic mirror or beam splitter and is brought to a point of focus by the objective lens at the level labeled ”Plane of Focus”. Fluorescence is emitted by the specimen from the point of focus (solid rays) and passes back through the objective lens, through the dichroic mirror and confocal aperture to the detector. However, fluorescence will also be emitted from planes above and below the plane of focus (dotted and dashed lines), but this light will be prevented from reaching the detector by the confocal aperture. To collect a two-dimensional (2D) image, the laser beam is usually scanned across the specimen in a ”rastor” pattern by a pair of mirrors (not drawn). To generate serial optical sections, the plane of focus is moved in the Z direction after each 2D image is collected. The net result is that a confocal microscope can usually produce thin, optical sections with greater resolution and contrast and with greater sensitivity than conventional, wide-field microscopes. This means that confocal microscopy can allow the assessment of tissue structures within thick specimens using non-invasive optical sectioning techniques, such that fixation and sectioning of specimens is no longer mandatory. Given that tissue fixation is often not required for the purposes of confocal fluorescence imaging, it is now possible to image events within and around living cells in various preparations including thick tissue slices. In addition, images can be obtained up to 200 mm into the outer layer of whole organs using serial optical sections that can be rapidly processed by computer graphics programs into three dimensional (3D) reconstructions. Confocal microscopy can improve lateral (x,y) resolution to approximately 200 nm (compared to approximately 400 nm in wide field microscopy). Additionally, image resolution in the z (vertical) axis is also improved (approximately 500 nm). This improvement is at least partially obtained through rejection of out-of-focus light, particularly in thick tissue sections or in living specimens (1,3). Figure 1 is a schematic representation of the light paths involved in obtaining a confocal image. Out-of-focus light originating from the specimen is blocked by an aperture before it reaches the detector. Only light originating from the plane of focus is used to create the image, i.e., an optical section (Fig. 2a,b). Additional (serial) in-focus optical sections can be produced by changing the focus of the microscope in the z-axis to produce a z-series. This can then be written to a computer and manipulated to produce an extended focus image (projection) with great depth of field (Fig. 2c). Figure 2. Confocal images taken from a ”whole mount” of rat tracheal tissue stained with an antibody to the neuronal protein marker PGP 9.5. The primary antibody has subsequently been localized with a second antibody labeled with fluorescein isothiocyanate (FITC). A 60 mm ”stack” or Z-series of images was collected at 2 mm intervals using a Bio-Rad MRC-1000/1024 UV confocal microscope. Single optical sections were taken (A) 18 mm and (B) 56 mm below the tissue surface. (C) A maximum intensity projection (extended focus image) of the entire data set. This projection method is most simply explained by considering each image of the original data set being mounted on individual transparent sheets. An image similar to the computer generated projection could be produced by shining a light through the transparent sheets and ”projecting” an image onto a wall. Scale bars = 50 µm. Confocal fluorescence microscopy is ideal for the rapid assessment of the distribution and localization of extracellular and intracellular macro- molecules, including proteins and nucleic acids, as well as intracellular ions such as calcium. The production of optical tissue sections in a few minutes, rather than the production of expensive and time-consuming slide-mounted tissue sections, means that the original tissue sample remains intact and can thus be repeatedly examined. Such sections can be rapidly combined to provide 3D tissue reconstructions of multiple cellular or extracellular entities. Extracellular tissue elements, surface, and intracellular profiles can be obtained following the use of multiple fluorescently tagged antibodies targeting specific antigens. For example, cell type-specific proteins such as neuronal protein gene product 9.5 (PGP 9.5) can be labeled with anti-PGP 9.5 antibodies. Similarly,
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