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

Multiphoton Fluorescence Microscopy
Interactive Tutorials

Excitation Photobleaching Patterns

Multiphoton fluorescence microscopy utilizes diffraction-limited focusing by a high numerical aperture objective to localize the spatial concentration of excitation light to narrow region near the focal point. This tutorial compares excitation-induced photobleaching patterns that occur near the focal region in both multiphoton and confocal microscopy systems.

The tutorial initializes with an x-z profile cross section of a thick film containing rhodamine-impregnated formvar sandwiched between a microscope coverslip and glass slide appearing in the applet window. A virtual raster scanning pattern traverses the long axis of the sandwiched film to simulate excitation by either multiphoton or confocal laser illumination sources. Radio buttons labeled Two-Photon and Confocal are used to toggle between the two excitation modes. The Laser Scan Speed slider can be adjusted to either increase or decrease the specimen scanning speed, and the Photobleach Rate slider will perform the same function with chromophore secondary fluorescence bleaching rates. A check box labeled Focal Point can be turned on to reveal a white outline of the focal region in both the two-photon and confocal microscopy modes.

Absorption of two-photon excitation is a non-linear optical effect with a probability distribution that drops as the fourth power of the distance along the optical axis (denoted the z-axis) outside of the focal region. When illuminating a uniform distribution of chromophores with multiphoton excitation (as depicted in this tutorial), 80 percent of the absorbed photons occur in a well-defined focal volume that is manifested by an ellipsoid having dimensions of about 0.3 microns for the lateral axis and 1 micron in the axial direction. These dimensions were measured at a wavelength of 700 nanometers and an objective numerical aperture of 1.4. Three dimensional resolution in multiphoton microscopy is thus due entirely to the confinement of chromophore excitation to the focal volume, precluding out-of-focus photobleaching and resulting photodamage that occurs with confocal laser excitation. Another advantage of multiphoton microscopy is the lack of excitation attenuation by out-of-focus adsorption by chromophores residing in planes removed from the focal region.

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.

John C. Long and 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.
© 1995-2022 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, software, 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 12:00 PM
Access Count Since November 20, 2000: 43033
For more information on microscope manufacturers,
use the buttons below to navigate to their websites: