Theory of Confocal Microscopy

Laser scanning confocal microscopy represents one of the most significant advances in optical microscopy ever developed, primarily because the technique enables visualization deep within both living and fixed cells and tissues and affords the ability to collect sharply defined optical sections from which three-dimensional renderings can be created. The principles and techniques of confocal microscopy are becoming increasingly available to individual researchers as new single-laboratory microscopes are introduced. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

Introduction to Confocal Microscopy - 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 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 fluorescence microscopy, and the growing number of applications in cell biology that rely on imaging both fixed and living cells and tissues. In fact, confocal technology is proving to be one of the most important advances ever achieved in optical microscopy.

Fluorescence Excitation and Emission Fundamentals - Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories, fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime.

Fluorophores for Confocal Microscopy - Biological laser scanning confocal microscopy relies heavily on fluorescence as an imaging mode, primarily due to the high degree of sensitivity afforded by the technique coupled with the ability to specifically target structural components and dynamic processes in chemically fixed as well as living cells and tissues. Many fluorescent probes are constructed around synthetic aromatic organic chemicals designed to bind with a biological macromolecule (for example, a protein or nucleic acid) or to localize within a specific structural region, such as the cytoskeleton, mitochondria, Golgi apparatus, endoplasmic reticulum, and nucleus. Other probes are employed to monitor dynamic processes and localized environmental variables, including concentrations of inorganic metallic ions, pH, reactive oxygen species, and membrane potential. Fluorescent dyes are also useful in monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. In addition, fluorescent probes have been widely applied to genetic mapping and chromosome analysis in the field of molecular genetics.

Interference Filters for Fluorescence Microscopy - The performance of high-resolution fluorescence microscopy imaging systems and related quantitative applications, especially as applied in living cell and tissue studies, requires precise optimization of fluorescence excitation and detection strategies. Fluorescence microscopy techniques could not have advanced so dramatically in recent years without significant developments in every dimension of the current state of the art, including the optical microscopes, the biology and chemistry of fluorophores, and perhaps most important, filter technology. The utilization of highly specialized and advanced thin film interference filters has enhanced the versatility and scope of fluorescence techniques, far beyond the capabilities afforded by the earlier use of gelatin and glass filters relying on the absorption properties of embedded dyes.

Spectral Bleed-Through Artifacts in Confocal Microscopy - The spectral bleed-through of fluorescence emission (often termed crossover or crosstalk), which occurs due to the very broad bandwidths and asymmetrical spectral profiles exhibited by many of the common fluorophores, is a fundamental problem that must be addressed in both widefield and laser scanning confocal fluorescence microscopy. The phenomenon is usually manifested by the emission of one fluorophore being detected in the photomultiplier channel or through the filter combination reserved for a second fluorophore. Bleed-through artifacts often complicate the interpretation of experimental results, particularly if subcellular co-localization of fluorophores is under investigation or quantitative measurements are necessary, such as in resonance energy transfer (FRET) and photobleaching (FRAP) studies.

Resolution and Contrast in Confocal Microscopy - All optical microscopes, including conventional widefield, confocal, and two-photon instruments are limited by fundamental physical factors in the resolution that they can achieve. In a perfect optical system, resolution is limited by numerical aperture of the optical components and by the wavelength of the light, both incident and detected. The concept of resolution is inseparable from contrast, and is defined as the minimum separation between two points that results in a certain contrast between them. In a real fluorescence microscope, contrast is determined by the number of photons collected from the specimen, the dynamic range of the signal, optical aberrations of the imaging system, and the number of picture elements (pixels) per unit area.

Introduction to Lasers - Ordinary natural and artificial light is released by energy changes on the atomic and molecular level that occur without any outside intervention. A second type of light exists, however, and occurs when an atom or molecule retains its excess energy until stimulated to emit the energy in the form of light. Lasers are designed to produce and amplify this stimulated form of light into intense and focused beams. The word laser was coined as an acronym for Light Amplification by the Stimulated Emission of Radiation. The special nature of laser light has made laser technology a vital tool in nearly every aspect of everyday life including communications, entertainment, manufacturing, and medicine.

Laser Systems for Confocal Microscopy - The lasers commonly employed in laser scanning confocal microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.

Acousto-Optic Tunable Filters (AOTFs) - Several benefits of the AOTF combine to greatly enhance the versatility of the latest generation of confocal instruments, and these devices are becoming increasing popular for control of excitation wavelength ranges and intensity. The primary characteristic that facilitates nearly every advantage of the AOTF is its capability to allow the microscopist control of the intensity and/or illumination wavelength on a pixel-by-pixel basis while maintaining a high scan rate. This single feature translates into a wide variety of useful analytical microscopy tools, which are even further enhanced in flexibility when laser illumination is employed.

Non-Coherent Light Sources for Confocal Microscopy - The traditional illumination system in the modern widefield microscope utilizes a tungsten-halogen source for transmitted light and a short-arc lamp for fluorescence excitation. Various lasers have been utilized as a light source for widefield observation by a few investigators, but the advent of the confocal microscope vastly increased laser use in microscopy. This discussion reviews the merits and limitations of non-coherent (or non-laser) light sources in confocal microscopy, both as light sources for confocal illumination and as secondary sources for widefield microscopy in confocal microscopes. Two initial issues frequently arise when illumination systems for confocal microscopes are considered, and these have a direct bearing on the choice of light sources for a particular instrument.

Confocal Microscope Objectives - For any conventional optical microscope configuration, the objective is the most critical component of the system in determining the information content of the image. The contrast and resolution of fine specimen detail, the depth within the specimen from which information can be obtained, and the lateral extent of the image field are all determined by the design of the objective and its performance under the specific conditions employed for the observation. Additional demands are imposed on the objective in scanning confocal techniques, in which this crucial imaging component also serves as the illumination condenser and is often required to perform with high precision at a wide range of wavelengths and at very low light levels without introducing unacceptable image-degrading noise.

Confocal Microscope Scanning Systems - Confocal imaging relies upon the sequential collection of light from spatially filtered individual specimen points, followed by electronic signal processing and ultimately, the visual display as corresponding image points. The point-by-point signal collection process requires a mechanism for scanning the focused illuminating beam through the specimen volume under observation. Three principal scanning variations are commonly employed to produce confocal microscope images. Fundamentally equivalent confocal operation can be achieved by employing a laterally translating specimen stage coupled to a stationary illuminating light beam (stage scanning), a scanned light beam with a stationary stage (beam scanning), or by maintaining both the stage and light source stationary while scanning the specimen with an array of light points transmitted through apertures in a spinning Nipkow disk. Each technique has performance features that make it advantageous for specific confocal applications, but that limit the usefulness in others.

Signal-to-Noise Considerations - In any quantitative assessment of imaging capabilities utilizing digital microscopy techniques, including confocal methods, the effect of signal sampling on contrast and resolution must be considered. The measured signal level values do not directly represent the number of photons emitted or scattered by the specimen, but are proportional to that number. Furthermore, each individual sample of signal intensity is only an approximation of the number of collected photons, and will vary with repeated measurement. The variation, referred to as noise, imparts an uncertainty in the quantification of intensity, and therefore in the contrast and resolution of the image data.

Electronic Light Detectors: Photomultipliers - In modern widefield fluorescence and laser scanning confocal optical microscopy, the collection and measurement of secondary emission gathered by the objective can be accomplished by several classes of photosensitive detectors, including photomultipliers, photodiodes, and solid-state charge-coupled devices (CCDs). In confocal microscopy, fluorescence emission is directed through a pinhole aperture positioned near the image plane to exclude light from fluorescent structures located away from the objective focal plane, thus reducing the amount of light available for image formation. As a result, the exceedingly low light levels most often encountered in confocal microscopy necessitate the use of highly sensitive photon detectors that do not require spatial discrimination, but instead respond very quickly with a high level of sensitivity to a continuous flux of varying light intensity.

Electronic Imaging Detectors - Over the past several years, the rapidly growing field of fluorescence microscopy has evolved from a dependence on traditional photomicrography using emulsion-based film to one in which electronic images are the output of choice. The imaging device is one of the most critical components in fluorescence microscopy because it determines at what level specimen fluorescence may be detected, the relevant structures resolved, and/or the dynamics of a process visualized and recorded.

Fluorochrome Data Table - As a guide to fluorophores for confocal and widefield fluorescence microscopy, the table presented in this section lists many commonly-used fluorochromes, with their respective peak absorption and emission wavelengths and suggested laser illumination sources. While the authors assume responsibility for the accurate reporting of the data as published in various reliable sources, several caveats must be given. Fluorochrome dyes are environmentally sensitive and different results will be obtained with different solvents and applications. In reviewing the literature, one will frequently find somewhat different data supplied for the identical fluorochrome. There are also, in many instances, several sub-varieties of a fluorochrome.

Glossary of Terms in Confocal Microscopy - The complex nomenclature of fluorescence microscopy is often confusing to both beginning students and seasoned research microscopists alike. This resource is provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in fluorescence and laser scanning confocal microscopy.

Recommended Books on Confocal Microscopy - A surprisingly limited number of books dealing with various aspects of laser scanning and spinning disk confocal microscopy and related techniques are currently available from the booksellers. This section lists the FluoView Resource Center website development team's top 12 recommended books. Although the volumes listed in this section deal pricipally with confocal microscopy and related methodology, there exist a number of additional books that contain focused treatments of the materials described below, and these should also be consulted for specific techniques and timely review articles.

Basic Concepts in Laser Scanning Confocal Microscopy (PDF; 2.8 Mb) - 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. Download this review article to learn more.

Interactive Java Tutorials - Explanations for many of the exceedingly complex concepts in laser scanning confocal microscopy can significantly benefit from the assistance of interactive tutorials that enable the student to obtain instanteous (real-time) response to changes in variables. The tutorials in section address the basic aspects of confocal microscopy instrumentation, laser systems, detectors, image processing, resolution, contrast, and many other aspects of the technique. All interactive Java tutorials require the Java Virtual Machine, which is available without cost as a browser plug-in from Sun Microsystems.