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.