Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest.

Basic Concepts - Current instruments are highly evolved from the earliest versions, but the principle of confocal imaging advanced by Marvin Minsky, and patented in 1957, is employed in all modern confocal microscopes. In a conventional widefield microscope, the entire specimen is bathed in light from a mercury or xenon source, and the image can be viewed directly by eye or projected onto an image capture device or photographic film. In contrast, the method of image formation in a confocal microscope is fundamentally different. Illumination is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen. This point of illumination is brought to focus in the specimen by the objective lens, and laterally scanned using some form of scanning device under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube (PMT) through a pinhole (or in some cases, a slit), and the output from the PMT is built into an image and displayed by the computer. Although unstained specimens can be viewed using light reflected back from the specimen, they usually are labeled with one or more fluorescent probes.

Imaging Modes - A number of different imaging modes are used in the application of confocal microscopy to a vast variety of specimen types. They all rely on the ability of the technique to produce high-resolution images, termed optical sections, in sequence through relatively thick sections or whole-mount specimens. Based on the optical section as the basic image unit, data can be collected from fixed and stained specimens in single, double, triple, or multiple-wavelength illumination modes, and the images collected with the various illumination and labeling strategies will be in register with each other. Live cell imaging and time-lapse sequences are possible, and digital image processing methods applied to sequences of images allow z-series and three-dimensional representation of specimens, as well as the time-sequence presentation of 3D data as four-dimensional imaging. Reflected light imaging was the mode used in early confocal instruments, but any of the transmitted light imaging modes commonly employed in microscopy can be utilized in the laser scanning confocal microscope.

Specimen Preparation and Imaging - The procedures for preparing and imaging specimens in the confocal microscope are largely derived from those that have been developed over many years for use with the conventional wide field microscope. In the biomedical sciences, a major application of confocal microscopy involves imaging either fixed or living cells and tissues that have usually been labeled with one or more fluorescent probes. A large number of fluorescent probes are available that, when incorporated in relatively simple protocols, specifically stain certain cellular organelles and structures. Among the plethora of available probes are dyes that label nuclei, the Golgi apparatus, the endoplasmic reticulum, and mitochondria, and also dyes such as fluorescently labeled phalloidins that target polymerized actin in cells. Regardless of the specimen preparation protocol employed, a primary benefit of the manner in which confocal microscopy is carried out is the flexibility in image display and analysis that results from the simultaneous collection of multiple images, in digital form, into a computer.

Critical Aspects of Confocal Microscopy - Quantitative three-dimensional imaging in fluorescence microscopy is often complicated by artifacts due to specimen preparation, controllable and uncontrollable experimental variables, or configuration problems with the microscope. This article, written by Dr. James B. Pawley, catalogs the most common extraneous factors that often serve to obscure results collected in fluorescence widefield and confocal microscopy. Among the topics discussed are the laser system, optical component alignment, objective magnification, bleaching artifacts, aberrations, immersion oil, coverslip thickness, quantum efficiency, and the specimen embedding medium.

Aberrations in Multicolor Confocal Microscopy - Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in widefield microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.

Three-Color Imaging for Confocal Microscopy - The laser scanning confocal microscope (LSCM) is routinely used to produce digital images of single-, double-, and triple-labeled fluorescent samples. The use of red, green and blue (RGB) color is most informative for displaying the distribution of up to three fluorescent probes labeling a cell, where any co-localization is observed as a different additive color when the images are colorized and merged into a single three-color image. In this section we present a simplified version of a previously published method for producing three-color confocal images using the popular image manipulation program, Adobe Photoshop. In addition, several applications of the three-color merging protocol for displaying confocal images are discussed. Note that these digital methods are not confined to images produced using the LSCM and can be applied to digital images imported into Photoshop from many different sources.

Basics of Confocal Reflection Microscopy - Confocal reflection microscopy can be utilized to gather additional information from a specimen with relatively little extra effort, since the technique requires minimum specimen preparation and instrument re-configuration. In addition, information from unstained tissues is readily available with confocal reflection microscopy, as is data from tissues labeled with probes that reflect light. The method can also be utilized in combination with more common classical fluorescence techniques. Examples of the latter application are detection of unlabeled cells in a population of fluorescently labeled cells and for imaging the interactions between fluorescently labeled cells growing on opaque, patterned substrata.

Confocal Microscopy Image Gallery - The Nikon MicroscopyU Confocal Image Gallery features digital image sequences captured using a Nikon PCM-2000 confocal microscope scanning system coupled to an Eclipse E-600 upright microscope. Successive serial optical sections were recorded along the optical axis of the microscope over a range of specimen planes. These sequences are presented as interactive Java tutorials that allow the visitor to either "play" the series of sections automatically, or to utilize a slider to scroll back and forth through the images.

Laser Scanning Confocal Microscopy - (approximately a 30 second download on 28.8K modems) Several methods have been developed to overcome the poor contrast inherent with imaging thick specimens in a conventional microscope. Specimens having a moderate degree of thickness (5 to 15 microns) will produce dramatically improved images with either with confocal or deconvolution techniques. The thickest specimens (20 microns and above) will suffer from a tremendous amount of extraneous light in out-of-focus regions, and are probably best-imaged using confocal techniques. This tutorial explores imaging specimens through serial z-axis optical sections utilizing a virtual confocal microscope.

Reflected Confocal Microscopy: Integrated Circuit Inspection - Examine individual layers on the surface of integrated circuits with this interactive tutorial. Digital images for the tutorial were collected with a Nikon Optiphot C200 reflected light confocal microscope. For each sequence, a series of z-axis optical sections was recorded as the microscope was successively focused (at 1-micrometer steps) deeper within the patchwork of circuitry on the surface of the silicon chips.