Microscopy Primer
Light and Color
Microscope Basics
Special Techniques
Digital Imaging
Confocal Microscopy
Live-Cell Imaging
Microscopy Museum
Virtual Microscopy
Web Resources
License Info
Image Use
Custom Photos
Site Info
Contact Us

The Galleries:

Photo Gallery
Silicon Zoo
Chip Shots
DNA Gallery
Amino Acids
Religion Collection
Cocktail Collection
Screen Savers
Win Wallpaper
Mac Wallpaper
Movie Gallery

Fluorescence Microscopy

Combination Methods with Phase Contrast

To minimize the effects of photobleaching, fluorescence microscopy can be combined with other techniques that are non-destructive to the fluorochrome, such as differential interference contrast (DIC), Hoffman modulation contrast (HMC), transmitted darkfield illumination, and phase contrast.

The idea is to locate a specific area of interest in a specimen using the non-destructive contrast enhancing technique then, without relocating the specimen, switch the microscope to fluorescence mode. The results of a typical experiment of this type are illustrated in Figure 1. Figure 1(a) illustrates 3T3 fibroblasts in monolayer tissue culture imaged using phase contrast optics. The cell line was established from a National Institutes of Health line of Swiss mouse embryo cells, which are highly contact inhibited and useful for studies involving sarcoma virus formation and leukemia virus propagation. The photomicrograph in Figure 1(b) shows the same viewfield, but this time imaged using fluorescence illumination (a mercury vapor lamp and an Olympus WU filter cube) with cells stained by the fluorochrome 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid specific dye with an emission maximum at 461 nanometers, which is used to selectively stain nuclei and chromatin. Figure 1(c) illustrates the two techniques used in combination to produce a beautiful photomicrograph of fluorescent-stained 3T3 cellular nuclei superimposed on a phase contrast image of the fibroblast cell membranes and internal organelles. This image was recorded with a specialized fluorite objective designed with phase rings to permit simultaneous observation of both fluorescence and phase contrast with the same objective.

The microscope configuration typically utilized to simultaneously image specimens using both phase contrast and fluorescence illumination is illustrated in Figure 2. Phase contrast is conducted using transmitted light provided by a tungsten-halogen lamp positioned in a lamphouse attached to the microscope base. Light passing through the field diaphragm is reflected by a mirror into the substage condenser and through a phase annulus of the proper dimensions.

Interactive Tutorial
Fluorescence Combination Microscopy
Explore combinations of fluorescence microscopy with additional contrast-enhancing techniques using both phase contrast and differential interference contrast (DIC) methods. 

Light diffracted by the specimen is first passed through a phase plate positioned in the objective rear focal plane before interfering at the intermediate image plane with light passing through the specimen undiffracted. Simultaneously, ultraviolet light emitted by a mercury burner is passed through an exciter filter, then reflected by a dichroic mirror onto the specimen from above. Secondary fluorescence emitted by the chromophore attached to the stained specimen is captured by the objective and passed through the barrier filter and into the eyepieces and/or phototube. This configuration can be used to image specimens using the techniques (fluorescence and phase contrast) individually or in combination.

Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.



Questions or comments? Send us an email.
© 1998-2022 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, scripts, or applets may be reproduced or used in any manner without permission from the copyright holders. Use of this website means you agree to all of the Legal Terms and Conditions set forth by the owners.
This website is maintained by our
Graphics & Web Programming Team
in collaboration with Optical Microscopy at the
National High Magnetic Field Laboratory.
Last modification: Tuesday, Sep 11, 2018 at 11:09 AM
Access Count Since July 30, 1998: 80642
For more information on microscope manufacturers,
use the buttons below to navigate to their websites: