Spectral bleed-through of fluorescence emission occurs due to the broad bandwidths and asymmetrical spectral profiles exhibited by fluorophores, can be a problem in laser scanning confocal fluorescence microscopy.

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passes wavelengths between 535 and 575 nm (Fluores- cein) and 575-655 nm (Cy3). For the Cy5 the 633 nm wavelength line of a HeNe laser was used for excita- tion ...

Explore the matching of dual fluorophores with efficient laser excitation lines and determination of the bleed-through level that can be expected as a function of the detection window wavelength profiles in this tutorial.

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.

Confocal microscopy has advantages over widefield optical microscopy, including the ability to eliminate or reduce background information away from the focal plane and collect serial optical sections from thick specimens.

Fluorescence is a member of the luminescence family of processes in which molecules emit light from electronically excited states created by either a physical, mechanical (friction), or chemical mechanism.

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Applications available to laser scanning confocal microscopy includes a variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues.

Biological laser scanning confocal microscopy relies on fluorescence as an imaging mode due to the high degree of sensitivity afforded by the technique coupled with the ability to specifically target structural components.

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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 objective.

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Three principal scanning variations are employed to produce confocal microscope images. Each technique has features that make it advantageous for specific confocal applications, but limit the usefulness in others.

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. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, spectral technology, and fluorophores for highly specific targets.

In a fluorescence microscope, contrast is determined by the number of photons collected, the dynamic range of the signal, optical aberrations of the imaging system, and the number of picture elements (pixels) per unit area.

A CPL (circular polarizing filter or polarizer for short) is a very effective tool for removing reflections and glare from water and other reflective surfaces.

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In laser scanning confocal microscopy, the collection of secondary emission gathered by the objective can be accomplished by classes of photosensitive detectors, including photomultipliers, photodiodes, and CCDs.

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.

Featured here are resources 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.

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Discover and explore the gallery of various interactive Java tutorials designed to explain and aid students visually in understanding complex and difficult concepts in confocal microscopy.

As a guide to fluorophores for confocal and widefield fluorescence microscopy, the table presented lists many commonly-used fluorochromes, with their respective peak absorption, and emission wavelengths.

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Several benefits of the AOTF combine to enhance the versatility of the latest generation of confocal instruments, and these devices are becoming popular for control of excitation wavelength ranges and intensity.

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Laser scanning confocal microscopy (LSCM) is a tool that has been extensively utilized for inspection of semiconductors, is now becoming a mainstream application in cell biology. The links provided in this section from the Olympus Microscopy Resource Center web site offer tutorials, instrumentation, application notes, technical support, glossaries, and reference materials on confocal microscopy and related techniques.

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by B Huang · 2010 · Cited by 1389 — The diffraction-limited image resolution of objective lens with a high numerical aperture is ~250 nm perpendicular to the direction of light propagation (i.e., ...

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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 utilization of specialized and advanced thin film, interference filters have enhanced the versatility of fluorescence techniques, far beyond the capabilities of the earlier use of gelatin and glass filters.

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The lasers used in laser scanning confocal microscopy are high-intensity monochromatic light sources, which are useful for techniques including optical trapping, lifetime imaging studies, and total internal reflection fluorescence.

Reviewed in this article are the merits and limitations of non-coherent light sources in confocal microscopy, both as light sources for confocal illumination and as secondary sources for widefield microscopy in confocal microscopes.

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