Scientists at Osaka University in Toyonaka and Suita, Japan, have developed a multiphoton fluorescence microscope that uses a Cr:forsterite laser as a near-infrared excitation source. They propose that such microscopes will be useful for the imaging of opaque, fragile biological specimens. The benefits of the Cr:forsterite (Cr4+:Mg2SiO4) laser in the setup derive from its femtosecond pulse duration and wavelength of 1.26 µm, explained Hiroshi Miyasaka, a professor and the head of the Miyasaka Lab at the university’s graduate school of engineering science. The short pulse width at the sample — approximately 35 fs — enables the excitation of low-frequency molecular vibrations for the study of ultrafast dynamics. It also enables high-order multiphoton processes, resulting in a high spatial resolution and making the source suitable for the stimulation of various fluorescent dyes with absorptions near 630, 420 and 315 nm.The near-infrared microscope uses a Cr:forsterite laser with pulse durations of 35 fs as an excitation source. A demonstration of the instrument yielded multiphoton fluorescence images of various microcrystals, including this sample of anthracene. Scale bar is 5 µm. Courtesy of Hiroshi Miyasaka.The relatively low pulse energy at the sample — on the order of 1 to 2 nJ — prevents thermal damage, and the near-IR output results in lower scattering in biological samples and, hence, greater imaging depths. Despite the advantages, Miyasaka said, commercial considerations have led Ti:sapphire sources to dominate in multiphoton fluorescence microscopy and have prevented the widespread use of Cr:forsterite lasers for this application.“Researchers who want to use such a laser system have to construct it by themselves,” he said.The scientists’ Cr:forsterite laser features a 19-mm-long crystal that is cooled to –10 °C and pumped with 7 W of 1064-nm CW radiation from a Spectra-Physics Nd:YVO4 laser to produce 1.2- to 1.35-µm radiation. A Bragg cell is used for cavity dumping, resulting in output pulse energies of 12 nJ at 100 kHz.The microscopy setup incorporates an Olympus Corp. inverted microscope equipped with a 100×, 0.95-NA objective, also from Olympus. The resulting fluorescence from the sample is directed to a Hamamatsu Photonics KK avalanche photodiode, an Ocean Optics Inc. fiber-coupled spectrometer or a Flovel Co. Ltd. CCD camera.In a demonstration of the instrument, the researchers collected two-, three- and four-photon fluorescence spectra and images of perylene, anthracene and pyrene microcrystals. Miyasaka said that subsequent experiments, the details of which are pending publication, quantified the spatial resolution of the setup using a photochemical reaction and that the scientists are investigating potential applications of higher-order multiphoton imaging with the microscope. Journal of Physical Chemistry B, Jan. 26, 2006, pp. 1091-1094.