Multiphoton Fluorescence Microscopy
Ti:Sapphire Mode-Locked Lasers
The self mode-locked Ti:sapphire pulsed laser is currently one of the preferred laser excitation sources in a majority of multiphoton fluorescence microscopy investigations. This tutorial explores the operation of Ti:sapphire lasers over a broad range of near-infrared wavelengths with variable pulse widths and an adjustable applet speed control.
The tutorial initializes with a single photon from the Ti:sapphire crystal being excited by the accompanying continuous wave argon-ion pump laser (not illustrated, but emitting blue-green photons through the input Brewster window). Light from the pump laser enters the Ti:sapphire laser housing and is reflected by a series of mirrors into a Ti:sapphire crystal in the laser cavity. Photons emitted by the doped sapphire crystal are guided through the resonator cavity by reflection from the surface of routing mirrors. Other elements in the 10-mirror folded cavity include rod focusing mirrors, an output coupler, a high reflector, beam folding mirrors, dispersion control prisms, and a tuning slit for adjusting the output wavelength. The folded beam is finally passed through an acousto-optic modulator (AOM), which ensures a constant frequency for mode-locked operation at laser start-up. Before final output, the laser beam passes through a beamsplitter where a portion of the light is diverted to a fast photodiode that is coupled to an electronic clocking circuit through a feedback loop. Finally, the beam travels through a Brewster output window to exit the laser cavity housing.
Several sliders control laser parameters that serve to modify how the applet functions. The Pulse Width slider can be utilized to adjust this value between 30 and 150 femtoseconds, and the Wavelength slider operates the tuning slit to vary output wavelengths from 690 to 1050 nanometers. A third slider controls the applet run speed.
Ti:sapphire mode-locked lasers provide a large wavelength tuning range, from about 690 to over 1050 nanometers, with pulse widths approximately 100 femtoseconds in length. Note that the lowest pulse width attainable in the actual Spectra Physics laser is 50 femtoseconds. In addition, these lasers have sufficient power (greater than 100 milliwatts throughout the tuning range) for saturation of two-photon excitation in most fluorophores. To ensure proper cooling and humidity control of the laser crystal, nitrogen gas is pumped into the sealed laser head, which is maintained at constant temperature by an external chiller.
The Titanium trivalent cation is responsible for laser action in doped sapphire crystals, which are produced by introducing titanium oxide into a melt of aluminum oxide. In the melt, some of the titanium ions diffuse into the aluminum oxide and are incorporated into lattice positions in place of aluminum cations. When excited by the argon-ion pump laser, titanium atoms in doped sapphire crystals absorb the greenish-blue light, which is subsequently released as coherent infrared light via stimulated emission. As the emitted light is repeatedly reflected back through the crystal with mirrors, it is amplified until laser oscillation occurs. The absorption spectrum for titanium-doped sapphire is illustrated in Figure 1, and extends from less than 400 nanometers to about 650 nanometers. Simultaneously plotted in the same figure is the emission spectrum for the doped crystal.
The fluorescence emission spectrum for Ti:sapphire extends from 600 to over 1050 nanometers, covering the long-wavelength visible and short-wavelength near-infrared spectral regions. Lasing is only possible at wavelengths longer than about 675 nanometers because of overlap between the absorption and emission spectra in the 600 to 650 nanometer region. Other factors that limit the useful wavelength tuning range are mirror coatings, absorption of water from the atmosphere on reflecting surfaces, and tuning element losses.
Ti:sapphire lasers require a supporting pump laser of sufficient power to excite the crystal and initiate laser action. Power requirements vary with output demand, but strongly focused argon-ion lasers having values between 5 and 20 watts can be utilized for this purpose. Recently, new solid state lasers have been developed that employ nonlinear conversion to double the output frequency of infrared lasers to produce lower wavelength green light. These lasers are far more efficient than argon-ion lasers and also have reduced power and cooling demands.
The doped sapphire laser crystal is unusual in that, unlike a majority of lasers, broad energy levels exhibited by the doping titanium ions allow for a wide spectrum of output wavelengths (see above). In order to select specific wavelengths for emission, Ti:sapphire lasers are equipped with either a birefringent tuner (picosecond versions) or a movable slit (femtosecond lasers) to allow emission over a very narrow (5-15 nanometers) wavelength range.
The Spectra Physics Tsunami® laser is designed with a ten-mirror folded cavity (see tutorial illustration) when configured for femtosecond operation. Care must exerted in folded-cavity laser design due to optical aberrations (such as astigmatism) that occur when focusing mirrors are utilized at angles other than normal incidence. Spectra Physics engineers have employed Brewster-angle correcting rods and carefully chosen reflection angles to help eliminate artifacts in the output beam. Also included in the design is an acousto-optic modulator (AOM), which employs a piezo-electric transducer to generate an acoustic wave that controls the phase between longitudinal modes in the laser cavity and ensures an 82-megahertz nominal mode-locked operation when the laser is started. The modulator is cut to intersect the beam at Brewster's angle and is driven by a radio frequency signal.
Mirrors designed for femtosecond Ti:sapphire lasers have high reflectivity, and are made with a series of dielectric thin films applied to the glass surface. Like interference filters and antireflection coatings, these dielectric layers have tightly controlled thicknesses that allow successive reflections from each layer to add through constructive interference. This ensures that reflection from the mirror surfaces exceeds 99 percent, but also restricts the wavelength range that each mirror is capable efficiently reflecting. As a result, in order to achieve the broad emission wavelength spectrum (690 to 1050 nanometers) capabilities of doped sapphire lasers, several sets of mirrors must be utilized for specific sets of wavelengths (see Table 1), somewhat limiting the versatility of the system and making it more difficult for novice users. Ti:sapphire lasers made by other manufacturers have similar mirror kits.
Spectra Physics Tsunami® Mirror Sets
Tuning in Ti:sapphire femtosecond lasers is accomplished through a prism sequence and a variable slit mechanism. The prisms create a region in the laser cavity where emission wavelengths are spatially spread, thus providing an opportunity to select a narrow bandwidth by strategic placement of the slit. Output wavelength ranges are restricted to a limited portion of the spread by changing the position of the slit, and the bandwidth is controlled by the physical slit width.
Group velocity dispersion (GVD) is a complex phenomenon that contributes to pulse broadening as photons propagate through the laser cavity. This effect causes the velocity of lower visible wavelengths (blue and green) to be reduced relative to longer wavelengths (red) when passing through optical components, resulting in lowered peak intensities. Ti:sapphire lasers often utilize prism pairs to produce a negative GVD that increases the velocity of shorter wavelengths in order to maintain narrow pulses and balance the laser output. The optical components of microscopes (objective glass and dielectric coatings) utilized in multiphoton fluorescence experiments can also be a source of GVD, and can contribute to pulse spreading, especially when pump lasers of limited power are employed.
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.
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