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Laser Scanning Confocal Microscopy
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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.

Confocal Microscope Simulators

Laser Scanning Confocal Microscope Simulator - Perhaps the most significant advance in optical microscopy during the past decade has been the refinement of mainstream laser scanning confocal microscope (LSCM) techniques using improved synthetic fluorescent probes and genetically engineered proteins, a wider spectrum of laser light sources coupled to highly accurate acousto-optic tunable filter control, and the combination of more advanced software packages with modern high-performance computers. This interactive tutorial explores multi-laser fluorescence and differential interference contrast (DIC) confocal imaging using the Olympus FluoView FV1000 confocal microscope software interface as a model.

Comparing Confocal and Widefield Fluorescence Microscopy - Confocal microscopy offers several distinct advantages over traditional widefield fluorescence 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 dimensions of the focal plane. This interactive tutorial explores and compares the differences between specimens when viewed in a confocal versus a widefield fluorescence microscope.

Spectral Bleed-Through (Crossover) in Confocal Microscopy - Bleed-through (often termed crossover) of fluorescence emission, due to the broad spectral profiles exhibited by common fluorophores, is a fundamental problem that must be addressed in both widefield and laser scanning confocal fluorescence microscopy. The phenomenon is most often manifested by the emission of one fluorophore being detected in the channel or through the filter combination reserved for a second fluorophore. This interactive tutorial explores spectral bleed-through in laser scanning confocal microscopy and the mechanisms available to reduce or eliminate the artifact.

Confocal Microscope Systems

Laser Confocal Microscopy Scanning Modes - The wide range of laser scanning modes available in modern confocal microscopy enable investigators to fine-tune acquisition strategies in order to optimize data collection for three-dimensional imaging, time-lapse analysis, and a host of other specialized applications. Among the common scanning modes featured by most commercial microscopes are point, line, free line, parallel plane, and rectangle scanning over one or more dimensions. This interactive tutorial examines various scanning mechanisms in confocal microscopy utilizing a cube-shaped virtual specimen.

Galvanometer-Based Confocal Scanning Systems - In order to generate a digital image from an extended specimen in laser scanning confocal microscopy, the focused beam is scanned laterally (in the x-y plane) across the specimen surface in a rectangular raster pattern. Modern instruments utilize a scanning mechanism based on two high-speed vibrating mirrors driven by galvanometer motors to produce the scanning pattern. This interactive tutorial explores how the scanning mirrors are coordinated to direct the laser beam into the objective, and then to reflect secondary fluorescence gathered from the specimen back through the optical train to the emission filter.

Scanning System Basics - The three basic requirements of a laser scanning confocal microscope system are to bring the laser illumination to a focal point on the specimen, scan a selected area of the surface in a raster pattern, and then gather only the secondary fluorescence that originates from the immediate region being excited by the focused laser beam. During scanning, the focal point size should exhibit even illumination and be maintained as small as possible, two requirements that necessitate the objective rear aperture being completely filled with light throughout the scanning cycle. This interactive tutorial examines how the galvanometer-driven mirrors and optical system of a typical confocal microscope are configured to enable the objective rear aperture to be continuously filled with light during the raster scanning operation.

Lasers for Confocal Microscopy

Argon-Ion Lasers - As a distinguished member of the common and well-explored family of ion lasers, the argon-ion laser operates in the visible and ultraviolet spectral regions by utilizing an ionized species of the noble gas argon. Argon-ion lasers function in continuous wave mode when plasma electrons within the gaseous discharge collide with the excited laser species to produce light.

Diode Lasers - Semiconductor diode lasers having sufficient power output to be useful in optical microscopy are now available from a host of manufacturers. In general these devices operate in the infrared region, but newer diode lasers operating at specific visible wavelengths are now available. Diode lasers coupled to internal optical systems that improve beam shape have sufficient power and stability to rival helium-neon lasers in many applications. This interactive tutorial explores the properties of typical diode lasers and how specialized anamorphic prisms can be utilized for beam expansion.

Helium-Cadmium Lasers - Helium-cadmium (He/Cd) lasers are finding an increasing number of important applications in confocal microscopy due to their three primary emission spectral lines (322, 354, and 442 nanometers) in the ultraviolet and blue-violet regions. The shortest wavelength (322 nanometers) requires specialized ultraviolet transparent optics and is seldom used in microscopy, but membrane probes (such as indo-1 and fura-2) can be efficiently excited with the 354-nanometer line. The blue-violet spectral line is useful for a host of common fluorophores and fluorescent proteins in single, double, or triple labeling experiments. This interactive tutorial explores a simplified model of the helium-cadmium laser cavity operation.

Helium-Neon Lasers - Helium-neon lasers are among the most widely utilized laser systems for a broad range of biomedical and industrial applications, and display the most superior Gaussian beam quality of any laser. These lasers are readily available at relatively low cost, have compact size dimensions, and exhibit a long operating life (often reaching 40,000 to 50,000 hours). The low power requirements, superior beam quality (virtually a pure Gaussian profile), and simple cooling requirements (convection) make helium-neon lasers the choice system for many confocal microscopes.

Krypton-Argon Lasers - Air-cooled lasers using krypton-argon mixtures have become popular in confocal microscopy when several illumination wavelengths are required for dual or multiple-fluorophore studies. Such mixed-gas lasers are only capable of producing stable output on major lines that are well separated in the wavelength spectrum. Of the three laser lines typically utilized for confocal microscopy, the 488-nanometer and 568-nanometer lines have approximately equal power (10 to 15 milliwatts), while the 647-nanometer line has about 50 percent more (15 to 25 milliwatts). This interactive tutorial simulates the three major spectral lines produced by an krypton-argon mixed-gas laser.

Acousto-Optic Tunable Filters - Wavelength selection is of fundamental importance in many arenas of the optical sciences, including fluorescence spectroscopy and confocal microscopy. Electro-optic devices, such as the acousto-optic tunable filter (AOTF), are increasingly being employed to modulate the wavelength and amplitude of illuminating laser light in the latest generation of confocal microscopes. These filters do not suffer from the mechanical constraints, speed limitations, image shift, and vibration associated with rotating filter wheels, and can easily accommodate several laser systems tuned to different output wavelengths. In addition, acousto-optic filters do not deteriorate when exposed to heat and intense light as do fluorescence interference filters.

Stimulated Emission in a Laser Cavity - The amplification of light by stimulated emission is a fundamental concept in the basic understanding of laser action. This interactive tutorial explores how laser amplification occurs starting from spontaneous emission of the first photon to saturation of the laser cavity and the establishment of a dynamic equilibrium state.

Pockels Cell Laser Modulators - All lasers are susceptible to noise introduced by their power supplies. Switching power supplies, which have become common because of their efficiency and small size, are particularly likely to introduce laser system ripple at frequencies ranging into the tens of kilohertz. Such interference, when it affects the light beam in confocal microscopy systems, can be especially troublesome to diagnose and remove. The beam intensity of continuous wave lasers can be stabilized by either electronic control of the tube current or through utilization of external components that modulate the light intensity. This interactive tutorial examines how the Pockels cell modulator operates to stabilize laser beam intensity.

Fluorophores for Confocal Microscopy

Fluorescent Probe Excitation Efficiency - The absorption and fluorescence emission spectral profiles of a fluorophore are two of the most important criteria that must be scrutinized when selecting probes for applications in laser scanning confocal microscopy. In addition to the wavelength range of the absorption and emission bands, the molar extinction coefficient for absorption and the quantum yield for fluorescence emission should be considered. At laser excitation levels that do not saturate the fluorophore, fluorescence intensity is directly proportional to the product of the extinction coefficient and the quantum yield. This interactive tutorial examines how this relationship can be utilized to match fluorophores with specific lasers for confocal microscopy.

Colocalization of Fluorophores in Confocal Microscopy - Two or more fluorescence emission signals can often overlap in digital images recorded by confocal microscopy due to their close proximity within the specimen. This effect is known as colocalization and usually occurs when fluorescently labeled molecules bind to targets that lie in very close or identical spatial positions. This interactive tutorial explores the quantitative analysis of colocalization in a wide spectrum of specimens that were specifically designed either to demonstrate the phenomenon, or to alternatively provide examples of fluorophore targets that lack any significant degree of colocalization.

Choosing Fluorophore Combinations for Confocal Microscopy - In planning multiple label fluorescence staining protocols for widefield and laser scanning confocal fluorescence microscopy experiments, the judicious choice of probes is paramount in obtaining the best target signal while simultaneously minimizing bleed-through artifacts. This interactive tutorial is designed to explore the matching of dual fluorophores with efficient laser excitation lines, calculation of emission spectral overlap values, and determination of the approximate bleed-through level that can be expected as a function of the detection window wavelength profiles.

Optical Highlighter Fluorescent Proteins - Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or that are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. This interactive tutorial explores the optical conversion of several useful highlighter probes and simulates how these proteins would be viewed in an actual confocal microscope.

Fluorescent Protein Fluorophore Maturation Mechanisms - Autocatalytic formation of the fluorophore (also referred to as a chromophore) within the shielded environment of the polypeptide backbone during fluorescent protein maturation follows a surprisingly unified mechanism, especially considering the diverse natural origins of these useful biological probes. Shortly after synthesis, most fluorescent proteins slowly mature through a multi-step process that consists of folding, initial fluorophore ring cyclization, and subsequent modifications of the fluorophore. The spectral properties of fluorescent proteins are dependent upon the structure of the fluorophore as well as the localized interactions of amino acid residues in the immediate vicinity, and in some cases, residues far removed from the fluorophore. The interactive tutorials in this section explore fluorophore formation in a wide variety of spectrally diverse fluorescent proteins deduced from crystallographic studies.

Resolution, Contrast, and Sampling in Confocal Microscopy

Airy Pattern Basics - The three-dimensional diffraction pattern formed by a circular aperture near the focal point in a well-corrected microscope is symmetrically periodic along the axis of the microscope as well as radially around the axis. When this diffraction pattern is sectioned in the focal plane, it is observed as the classical two-dimensional diffraction spectrum known as the Airy pattern. This tutorial explores how Airy pattern size changes with objective numerical aperture and the wavelength of illumination; it also simulates the close approach of two Airy patterns.

Digital Image Sampling Frequency - In order to match the optical and electronic resolution of a confocal microscope, a digital image should have a sufficient number of samples per horizontal line so that the display faithfully represents the original signal presented to the digitizing device. This interactive tutorial explores how variations in specimen sampling frequency affect the resolution of the final image.

Airy Patterns and the Rayleigh Criterion - Airy diffraction pattern sizes and their corresponding radial intensity distribution functions are sensitive to the combination of objective and condenser numerical apertures as well as the wavelength of illuminating light (when monochromatic light is used to illuminate the specimen). For a well-corrected objective with a uniform circular aperture, two adjacent points are just resolved when the centers of their Airy patterns are separated by a minimum distance (D) equal to the radius (r) of the central disk in the Airy pattern.

Airy Patterns and Resolution Criteria (3-D Version) - When the separation distance between adjacent Airy patterns is greater than the central disk radius, the sum of the intensities yields two individual peaks. As the disks approach each other, the separation distance will reach a value equal to the central disk radius, a condition known as the Rayleigh criterion. At even closer approach, the separation distance is less than the central disk radius and the sum of the two peaks merges into a single peak. In the latter instance, the two Airy patterns are said not to be resolved. This tutorial explores with a three-dimensional model how Airy disk sizes, at the limit of optical resolution, vary with changes in objective numerical aperture and illumination wavelength, and how these changes affect the resolution (r) and contrast of the objective with regard to the Rayleigh and Sparrow limits as two point images merge together.

Confocal Microscope Detectors

Side-On Photomultipliers - In the side-on photomultiplier tube design, photons impact an internal photocathode and eject electrons from the front face (as opposed to the rear side as in the end-on designs). These ejected photoelectrons have trajectories angled at the first dynode, which in turn emits a larger quantity of electrons angled at the second dynode (and so on). Incident light is detected through the curved side of the envelope in side-on photomultipliers. Due to their high performance ratings and low cost, side-on photomultipliers are the most widely used tubes for general photometric applications, such as spectrophotometry, fluorimetry, and confocal microscopy.

Channel Photomultipliers - Channel photomultipliers represent a new head-on monolithic design that incorporates a unique detector having a semitransparent photocathode deposited onto the inner surface of the entrance window. The photomultiplier features similar functionality to conventional units, but with dramatically increased sensitivity and high quantum efficiency. Individual photoelectrons released by the photocathode enter a narrow and curved semiconductive channel that serves in place of the traditional dynode chain. Each time an electron impacts an inner wall of the channel, multiple secondary electrons are emitted. This interactive tutorial explores how electrons are multiplied within the conductive chain of a channel photomultiplier.