Fluorescence Microscopy

Review Articles

• Introduction to Fluorescence Microscopy

  Learn the basic concepts of fluorescence, a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical, mechanical, or chemical mechanism.

• The Fluorescence Microscope

  Unlike other modes of optical microscopy based on macroscopic specimen features, such as birefringence, fluorescence microscopy is capable of imaging the distribution of a single molecular species based solely on the properties of fluorescence emission.

• Light Sources

  To generate enough excitation light intensity to provide secondary fluorescence emission capable of detection, powerful light sources are needed such as LED, mercury, and xenon arc (burner) lamps.

• Introduction to Laser Scanning Microscopes

  Learn all about laser scanning microscopes, including their advantages, disadvantages, and the types of images they can produce.

• Optimization and Troubleshooting

  Reviewed in this article are key features of fluorescence microscopy such as detecting fluorescent objects that can be faintly visible or very bright relative to the background, as well as common problems with microscope configuration.

• Imaging Detectors

  The featured discussion is intended to aid in understanding the basics of light detection and to provide a guide for selecting a suitable detector for specific applications in fluorescence microscopy.

• Introduction to Fluorophores

  Widefield fluorescence and laser scanning confocal microscopy rely on secondary fluorescence emission as an imaging mode, primarily due to the high degree of sensitivity afforded by the techniques.

• Optical Highlighter Fluorescent Proteins

  Optical highlighters generally display little or no initial fluorescence under excitation at the imaging wavelength but increase their fluorescence intensity after activation by irradiation at a different wavelength.

• Fluorescence Microscopy Errors

  Read more about how photomicrography under fluorescence illumination conditions can present a unique set of circumstances that may pose special problems for the microscopist.

• Practical Aspects of Fluorescence Filter Combinations

  A wide spectrum of filter cubes is available from most major manufacturers, who now produce filter sets capable of imaging most of the common fluorophores in use today.

• Glossary of Terms in Fluorescence and Confocal Microscopy

  The featured 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.

Advanced Techniques in Fluorescence Microscopy

• Introduction to Confocal Microscopy

  Confocal microscopy offers the ability to control depth of field, eliminate or reduce of background information away from the focal plane, and the capability to collect serial optical sections from thick specimens.

• Multiphoton Excitation Microscopy

  Multiphoton fluorescence microscopy is a powerful tool combining the techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution and 3D images of specimens.

• Fluorescence Resonance Energy Transfer (FRET)

  When the technique of fluorescence resonance energy transfer (FRET) is applied to optical microscopy, it permits determination of the approach between two molecules within several nanometers.

• Total Internal Reflection Fluorescence Microscopy

  Total internal reflection fluorescence microscopy (TIRFM) is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.

• Laser Systems for Optical Microscopy

  The lasers employed in optical microscopy are high-intensity monochromatic light sources, which are useful for many techniques including optical trapping, lifetime imaging studies, and photobleaching recovery.

• Fluorescence & DIC Combination Microscopy

  Fluorescence microscopy can be combined with contrast enhancing techniques such as differential interference contrast (DIC) illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC.

• Fluorescence and Phase Contrast Combination Microscopy

  To minimize photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.

• Fluorescence Microscopy Digital Image Gallery

  Specimens featured in this gallery contain a variety of discipline examples using specific fluorochrome stains and autofluorescence. Images were captured with either digital camera systems or classical photomicrography on film with Fujichrome Provia 35 millimeter transparency film.

Anatomy of the Fluorescence Microscope

• Olympus Upright Microscope

  Learn about the Olympus upright epifluorescence microscope equipped with a vertical illuminator that contains a turret of filter cubes and a fluorescence excitation light source.

• Olympus Inverted Microscope

  Microscopes with an inverted-style frame (Olympus IX70 inverted microscope) are designed for tissue culture applications and are capable of producing fluorescence illumination through an episcopic and optical pathway.

Interactive Java Tutorials

• Fluorescence Microscope Light Pathways

  Explore illumination pathways in an upright microscope. The microscope drawing presented in the tutorial illustrates a cut-away diagram of the Olympus upright microscope.

• Inverted Microscope Light Pathways

  Explore light pathways through an inverted tissue culture microscope equipped with for both diascopic (tungsten-halogen) and epifluorescence (mercury arc) illumination.

Selected Literature References

• Selected Literature References

  The field of fluorescence microscopy is experiencing a renaissance with the introduction of new techniques such as confocal, multiphoton, deconvolution, and total internal reflection, especially when coupled to advances in chromophore and fluorophore technology. The reference materials listed below were utilized in the construction of the Fluorescence section of the Molecular Expressions Microscopy Primer.

Related Olympus Products:

• Microscopes

• Fluorescence Microscopes