Nikon MicroscopyU | Fluorescence Microscopy

Concepts in Fluorescence Microscopy

In the rapidly expanding fields of cellular and molecular biology, widefield and confocal fluorescence illumination and observation is becoming one of the techniques of choice. These techniques, which are almost universally employed in both the medical and biological sciences, have spurred the development of more sophisticated microscopes and numerous fluorescence accessories.

Review Articles

Introduction to Fluorescence Microscopy
Basic equipment and techniques necessary for observing specimens in fluorescence.
Basics of FRET Microscopy
Using fluorescence to examine dynamic interactions between probes in living cells.
Fluorescence in situ Hybridization
Referred to as FISH, the technique is used primarily for chromosomal analysis.
Introduction to Fluorescent Proteins
Genetically-encoded fluorescent probes that are revolutionizing live-cell imaging.
Stereomicroscopy Fluorescence Illumination
Fluorescence Illuminators enable examination of large specimens in stereomicroscopy.
Optical System and Detector Requirements
Considerations are detector sensitivity, speed of image acquisition, and photobleaching.

Total Internal Reflection Fluorescence
TIRF restricts the excitation and detection of fluorophores to a thin region of the specimen.
Multiphoton Microscopy
Mode-locked pulsed lasers are used for deep tissue imaging and optical sectioning.
Nikon Fluorescence Filter Sets
Discussion of the properties of various fluorescence filter combinations.
Fluorescent Protein Imaging Parameters
Filter requirements, photobleaching, objective choice, highlighters, and multicolor imaging.
Confocal Microscopy
Confocal microscopy offers several advantages over conventional microscopy.
Laser Safety Basics
Lasers of sufficient energy can seriously damage unprotected eyes.

Interactive Java Tutorials

Fluorescence Filter Noise Terminator
Fluorescence filter block design that eliminates the possibility of residual stray light.
FRET with Fluorescent Proteins
Use this tool to determine the optimum fluorescent protein pairs for FRET.
Matching Fluorescent Probes to Nikon Filters
Determine optimum choices among current Nikon fluorescence filter blocks.
Laser Scanning Confocal Microscopy
A virtual microscope tutorial featuring a wide variety of specimens.
Balancing Arc Lamp Excitation
Excitation filter balancers can be used to fine-tune fluorophore excitation.
Blue Excitation Filter Combinations
Variations in specimen contrast with longpass and shortpass filters.
Blue-Violet Excitation Filter Combinations
Explore how filter bandpass variations affect specimen contrast.
Dual Excitation Filter Combinations
Specimen contrast with three different dual filter combinations.
Green Excitation Filter Combinations
Filter sets for orange and red fluorophores, including rhodamine derivatives.

Focus and Alignment of Arc Lamps
Explore alignment and focusing of the arc lamp in a mercury or xenon burner.
Stereomicroscopy Fluorescence
Light pathways in fluorescence stereomicroscopy.
Fluorescent Proteins for Dual Labeling
Interactive tutorial used to optimize pairing of two fluorescent proteins.
Filter Combinations for Fluorescent Proteins
Identification of critical filter parameters for imaging fluorescent proteins.
Stereomicroscopy Fluorescence
Explore the internal optical pathways of the Nikon SMZ1500 stereomicroscope
Triple Excitation Filter Combinations
Filter sets for DAPI, FITC, and TRITC or Texas Red.
Ultraviolet Excitation Filter Combinations
Examine specimen contrast with longpass and shortpass filter sets.
Violet Excitation Filter Combinations
Violet excitation is useful to examine cyan fluorescent proteins.
Yellow Excitation Filter Combinations
Specimen contrast variations with narrow and wide bandpass filter combinations.

Interactive Flash Tutorials

Matching Fluorescent Probes with Filters
Explore the various fluorophores that can be imaged with Nikon filter sets.
CCD Resolution for Optical Microscopy
Matching digital camera and microscope resolution.

Fluorescent Protein Biosensors
Descriptions of selected fluorescent protein biosensor mechanisms.
TE2000 Inverted Microscope Light Paths
Animated cut-away diagram of a modern inverted microscope illustrating the light paths.

Digital Image Galleries

Fluorescence Microscopy Digital Images
Cells and tissues examined with synthetic fluorophores in fluorescence microscopy.
Digital Eclipse Image Gallery
Explore the performance of Nikon's DXM-1200 camera using several contrast techniques.

Stereomicroscopy Fluorescence Images
Fluorescent specimens imaged using the Nikon SMZ-1500 stereomicroscope.
Confocal Microscopy Image Gallery
Enjoy the beauty of autofluorescence in thick sections of animal and plant tissues.

Digital Video Galleries

Swept Field Confocal Digital Video Gallery
Live-cell imaging using high speed confocal microscopy and fluorescent proteins.

Confocal Microscopy Digital Video Gallery
Single and dual-color fluorescence imaging in laser scanning confocal microscopy.

Selected Literature References

Fluorescence Microscopy
Examining specimens labeled with molecules that absorb light and emit fluorescence.
Confocal Microscopy
Imaging with laser excitation and pinhole detection of fluorescence.
Fluorophore Co-Localization
Detecting the presence of two or more molecules at the same physical location.
Fluorescent Proteins
Fluorescent proteins are currently the probes of choice for live-cell imaging.
Synthetic Fluorophores and Hybrid Tags
A number of fluorophores have been developed for imaging living and fixed cells.
Multiphoton Microscopy
Unique excitation scheme that reduces photobleaching and phototoxicity.
Deconvolution Microscopy
A powerful tool for three-dimensional analysis of complex biological specimens.
Spectral Imaging and Linear Unmixing
A powerful technique for co-localization, FRET, and removing autofluorescence.

Autofluorescence
Natural fluorescence in plants and animals that can also be introduced by fixatives.
Spinning Disk Microscopy
Excellent technique for high-speed imaging of living cells in real time with a CCD camera.
Fluorescence Resonance Energy Transfer
Probing molecular interactions in living cells using fluorescent proteins and fluorophores.
Fluorescent Protein Biosensors
Genetically-encoded FRET biosensors are useful in detecting a many biological events.
Fluorescence Recovery after Photobleaching
FRAP is used to determine the mobility of fluorescently tagged proteins in live cells.
Total Internal Reflection Microscopy
TIRFM is useful for probing cellular processes that occur near the membrane.
Single Molecule Fluorescence Microscopy
This technique is emerging as a powerful tool for superresolution imaging.
Fluorescent Speckle Microscopy
Stochastic but sparse labeling of macromolecules to track protein dynamics.

Contributing Authors

Michael E. Dailey - Department of Biological Sciences and Neuroscience Program, 369 Biology Building, University of Iowa, Iowa City, Iowa, 52242.

Daniel C. Focht - Bioptechs Inc., 3560 Beck Road, Butler, Pennsylvania, 16002.

Alexey Khodjakov and Conly L. Rieder - Wadsworth Center, New York State Department of Health, Albany, New York, 12201, and Marine Biological Laboratory, Woods Hole, Massachusetts, 02543.

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

George H. Patterson and Jennifer Lippincott-Schwartz - Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Jason R. Swedlow and Paul D. Andrews - Division of Gene Regulation and Expression, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland.

Jennifer C. Waters - Nikon Imaging Center, LHRRB Room 113C, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts, 02115.

Stephen T. Ross, Anna Scordato, and Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.

John D. Griffin, Nathan S. Claxton, Matthew J. Parry-Hill, Thomas J. Fellers, Kimberly M. Vogt, Ian D. Johnson, Shannon H. Neaves, Omar Alvarado, Lionel Parsons, Jr., Michael A. Sodders, Richard L. Ludlow, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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