Nikon MicroscopyU | Live-Cell Imaging

Introduction to Live-Cell Imaging Techniques

An increasing number of investigations are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology.

Review Articles

Maintaining Live Cells on the Microscope
A discussion of pH, atmosphere, temperature, media, osmolarity, buffers, and antibiotics.
Live-Cell Imaging Culture Chambers
Chambers must offer excellent optical properties while maintaining healthy cells.
Introduction to Fluorescent Proteins
Genetically-encoded fluorescent probes that are revolutionizing live-cell imaging.
Focus Drift in Live-Cell Imaging
Review software and hardware solutions for combating focus drift in time-lapse imaging.

Optical System and Detector Requirements
Considerations are detector sensitivity, speed of image acquisition, and specimen viability.
The Automatic Microscope
Discussion includes shutters, filter wheels, autofocus, light sources, and stage control.
Imaging Fluorescent Proteins
Filter requirements, photobleaching, objective choice, highlighters, and multicolor imaging.
Nikon Perfect Focus System (PFS)
Explore Nikon's high-speed solution for maintaining stable focus in live-cell imaging.

Interactive Tutorials

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.
Matching Camera to Microscope Resolution
Vary numerical aperture, magnification, and video coupler size to match camera resolution.
Fluorescent Proteins for Dual Labeling
Interactive tutorial used to optimize pairing of two fluorescent proteins.
DIC Microscope Component Alignment
Examine conoscopic and orthoscopic viewfields in DIC microscopy.
Comparison of Phase Contrast and DIC
Examples of the same specimen viewed in either phase contrast or DIC.
Phase Contrast Microscope Alignment
Learn how to align phase rings with the condenser in a phase contrast microscope.
Alignment for Köhler Illumination
Explore the basic concepts in the proper alignment of an optical microscope.
The Nikon Perfect Focus System (PFS)
Examine how the Nikon PFS system operates to maintain stable focus in live-cell imaging.

Focus and Alignment of Arc Lamps
Explore alignment and focusing of the arc lamp in a mercury or xenon burner.
Filter Combinations for Fluorescent Proteins
Identification of critical filter parameters for imaging fluorescent proteins.
Optical Sectioning with de Sénarmont DIC
At high numerical apertures, DIC can be used for optical sectioning.
Adjustment of Objective Correction Collars
Learn how to adjust an objective correction collar to minimize spherical aberration.
Spatial Resolution in Digital Imaging
Spatial resolution refers to the number of pixels utilized in construction of the image.
Proximity-Focused Image Intensifiers
Light amplification with a micro-channel plate and photocathode.
Laser Scanning Confocal Microscopy
A virtual microscope tutorial featuring a wide variety of specimens.
Phase Plate Effects on Specimen Contrast
Examine contrast variations induced by altering phase plate absorption properties.
Perfect Focus Offset System Mechanics
Learn how the offset lens system enables the operation of the Nikon PFS.

Digital Image Galleries

Fluorescence Microscopy Digital Images
Cells and tissues examined with synthetic fluorophores in fluorescence microscopy.
Confocal Microscopy Image Gallery
Enjoy the beauty of autofluorescence in thick sections of animal and plant tissues.

Phase Contrast Digital Image Gallery
Specimen viewfields examined using positive and negative phase contrast.
Phase Contrast and DIC Image Gallery
Compare specimen contrast using these complementary imaging techniques.

Digital Video Galleries

Live-Cell Imaging: Cell Motility
Explore cell motility in differential interference contrast (DIC) microscopy.
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.
Pond Life
Observe the activities of a wide variety of microscopic organisms.

Selected Literature References

Live-Cell Imaging Techniques
Basic concepts in maintaining and imaging living cells on the microscope stage.
Focus Drift in Optical Microscopy
Focus drift describes the inability to maintain focus over an extended period of time.
Fluorescent Proteins
Fluorescent proteins are currently the probes of choice for live-cell imaging.
Fluorescence Recovery after Photobleaching
FRAP is used to determine the mobility of fluorescently tagged proteins in live cells.
Chromophore-Assisted Light Inactivation
CALI is a useful technique for selectively deactivating target molecules.

Live-Cell Imaging Chambers
Chambers specially designed to keep cells alive and healthy on the microscope stage.
Electron-Multiplying CCD Cameras
EMCCDs are the detector system of choice for live-cell imaging in real-time applications.
Fluorescent Protein Biosensors
Genetically-encoded FRET biosensors are useful in detecting a many biological events.
Synthetic Fluorophores and Hybrid Tags
A number of fluorophores have been developed for imaging living and fixed cells.
FRET Microscopy with Spectral Imaging
Excellent for ratiometric analysis of photobleaching and spectral imaging FRET.

Selected Internet Resources

Motorized Microscope Accessories
Links to the websites for manufacturers and distributors of live-cell imaging equipment.

Live-Cell Imaging and Perfusion Chambers
Links to the manufacturers and distributors of specimen chambers for live-cell imaging.

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.

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.

Melpomeni Platani - Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany.

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.

Yu-li Wang - University of Massachusetts Medical School, 377 Plantation Street, Suite 327, Worcester, Massachusetts, 01605.

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

Joel S. Silfies and Edward G. Lieser - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.

Tony B. Gines, Alex B. Coker, Nathan S. Claxton, Scott G. Olenych, John D. Griffin, 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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