Excitation Region Events

In multiphoton fluorescence microscopy, excitation of fluorophores is localized to a narrow region (approximately a micron thick at high numerical aperture) encompassing the microscope focal point, thus eliminating background fluorescence and out-of-focus flare that typically limits the effective sensitivity in confocal microscopy.

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This interactive tutorial explores events occurring in the microscope focal region during specimen excitation using long wavelength visible and near infrared laser illumination. The tutorial initializes with a random set of fluorophores (open blue circles) appearing in the region surrounding a virtual focal point of a multiphoton excitation experiment. Several of the fluorophores are constrained by the applet to reside at the Focal Point, which is also designated the Region of Multiphoton Excitation. Two radio buttons, labeled Two Photon (tutorial default) and Three Photon are used to toggle excitation between nonlinear two-photon or three-photon optical absorption events.

The Laser slider is utilized to tune the wavelength of a virtual Ti:sapphire laser between 700 and 1100 nanometers. To initiate excitation, use the mouse cursor to click the blue Pulse button, which will trigger a laser pulse traveling slowly from the top of the applet window and passing through the focal region to the bottom of the window. After the pulse has been triggered, the Pulse button will turn red and deactivate while photons emitted by the laser are traversing the tutorial window. After the pulse has traveled from the top to the bottom of the tutorial window, the Pulse button will turn blue again and is then reactivated. Placing a checkmark in the Auto Pulse checkbox will enable a continuous sequence of laser pulses. The wavelength of photons emanating from the laser source and passing through the microscope optical train is adjustable using the Laser slider. When the tutorial initializes, the Laser slider is set to a wavelength of 900 nanometers, with a two-photon absorption event occurring at 450 nanometers (blue). The default fluorophore emission wavelength is 530 nanometers (green secondary emission).

Applet speed is controlled by the Pulse Speed slider, which slows the movement of photons through the tutorial when moved to the left and increases photon speed when translated to the right. The Stokes Shift slider can be utilized to adjust the wavelength difference between excitation and emission maxima of the virtual fluorophores, with a range between 10 and 150 nanometers. The excitation and emission wavelengths (selected by the Laser and Stokes Shift sliders) and their associated visible light colors are presented beneath the Laser slider.

When the Three Photon radio button is selected, the tutorial switches to a second multiphoton excitation mode in which three photons of the same wavelength interact with the fluorophore simultaneously to trigger fluorescence excitation. For example, when the Laser slider is set to 1050 nanometers, and the Pulse button is triggered, fluorophores are excited at 350 nanometers by simultaneous absorption of three 1050 nanometer photons and subsequently emit secondary fluorescence at a wavelength of 430 nanometers. In the three-photon mode, both two-photon and three-photon excitation can be simultaneously conducted by selecting the Multiple Fluorophores checkbox. When this option is chosen, two excitation wavelengths are utilized to excite the virtual fluorophores, depending upon whether an individual fluorophore absorbs two or three photons emitted by the laser. The excitation and emission wavelengths for a two-photon absorption event (when the applet is in three-photon mode) are presented beneath the Multiple Fluorophores checkbox.

A useful advantage of three-photon excitation is ability to excite fluorophores with absorption bands in deep ultraviolet region. For example, a laser pulse having a photon wavelength of 720 nanometers can be utilized to excite a fluorophore that normally absorbs light at 240 nanometers. This technique is advantageous when employing standard microscope optics that cannot efficiently transmit wavelengths below 300 nanometers.

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.