Three-dimensional visualization of living cells is only a femtosecond away with a small, low-cost, ultrafast laser developed by Allister Ferguson of the Department of Physics and Applied Physics and the Institute of Photonics at the University of Strathclyde (Glasgow, Scotland). The device emits around 850 nm in 70-fs pulses with an average power of 20 mW; the laser crystal is chromium-doped (Cr3+) LiSAF, and a specially designed saturable Bragg reflector (SBR) is used to modelock the laser. The SBR—first conceived by Wayne Knox of Lucent Technologies (Holmdel, NJ)—is similar to a dielectric mirror, says Ferguson, with a quantum well in the top layer on a gallium arsenide (GaAs) substrate (see Fig. 1).
Low-intensity light is absorbed by the quantum well, but higher-intensity light passes through the well and is reflected by the dielectric stack. Because the SBR is within the cavity, high-intensity light is reflected back into the cavity. The laser pulse grows progressively shorter, but the gain bandwidth prevents it from becoming too short. "What makes this unique is that it is all-solid-state," Ferguson says. The laser has a typical repetition rate of 100 MHz. The pump source is a visible-emitting aluminum gallium indium phosphide (AlGaInP) semiconductor laser.
Multiphoton imaging is a prime application for such a device, says Ferguson. Femtosecond-laser technology has triggered activity in the use of multiphoton-induced fluorescence imaging, which builds on the widely adopted technique of confocal fluorescence imaging. The main advantages of the approach are that it enables live biological samples to develop with minimum disturbance and allows deep penetration and detection in the sample while reducing bleaching effects.
Historically, fluorescence has been excited throughout the cone of illumination of the incident laser beam. Ferguson says that because fluorescence is collected only at the focus, the bulk of the excitation is wasted; this leads to the generation of toxic products, which modify, and eventually kill, the sample under observation. In addition, the fluorescent dyes undergo only a limited number of excitation and emission cycles before bleaching and giving rise to reduced signals.
Diagnostic imaging
Multiphoton excitation, such as two-photon absorption, eliminates these problems (see Fig. 2). The excitation wavelength is shifted to about 1000 nm, which is twice the wavelength of the absorption peak of the dye used. When a high-power pulsed laser with short picosecond, subpicosecond, or femtosecond pulses is used, mean power levels are moderate and do not damage the specimen. Also, by using such lasers, the photon density is sufficiently high that two photons can be absorbed by the dye simultaneously. Excitation, therefore, occurs only at the point of focus of the microscope, causing less toxicity and bleaching.
"Two-photon microscopy already has had a massive impact in biological sciences and is beginning to become widely adopted," says Ferguson. "Better still, the peak power available from femtosecond sources is capable of producing three-photon and higher-order processes." With three-photon excitation, three photons are absorbed simultaneously, effectively tripling the excitation energy. This suggests that infrared excitation could be used to image ultraviolet-excited dyes.
The technique was also applied to the investigation of microcircuitry in integrated circuits by multiphoton excitation of currents in the silicon substrate of the circuit. Currently, such multiple-photon excitation is possible only with expensive, bulky, high-power, subpicosecond lasers. The femtosecond, diode-pumped Cr3+:LiSAF laser developed at Strathclyde requires no cooling and is only 7 × 10 × 18 in. Although the laser is a prototype, Ferguson expects volume applications to drive the price down to $20,000-$30,000. The laser was developed as part of a collaboration with Bio-Rad Microscience (Hemel Hempstead, England), which is considering the commercial implications of the device.