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2006, 110, 1091-1094 Published on Web 12/27/2005
Development of Near-Infrared 35 fs Laser Microscope and Its Application to the Detection of Three- and Four-Photon Fluorescence of Organic Microcrystals Hirohisa Matsuda,† Yousuke Fujimoto,† Syoji Ito,† Yutaka Nagasawa,† Hiroshi Miyasaka,*,† Tsuyoshi Asahi,‡ and Hiroshi Masuhara‡ DiVision of Frontier Materials Science, Graduate School of Engineering Science and Research Center for Materials Science at Extreme Conditions, Osaka UniVersity, Toyonaka, Osaka 560-8531, Japan, and Department of Applied Physics, Graduate School of Engineering, Osaka UniVersity, Suita, Osaka 565-0871, Japan ReceiVed: October 25, 2005; In Final Form: December 11, 2005
Femtosecond near-infrared laser microscope was developed with a home-built cavity-dumped chromium: forsterite laser as a light source centered at 1.26 µm. Optimization of the pulse duration achieved 35 fs fwhm at the sample position of the microscope after passing through a 100× objective. This system was applied to the detection of multiphoton fluorescence of some organic microcrystals. Excitation intensity dependence and the interferometric autocorrelation detection of the fluorescence clearly demonstrated that simultaneous three- and four-photon absorption processes are responsible for the production of the excited state for perylene and anthracene microcrystals, respectively. The spatial resolution along the optical axis and its dependence on the order of the multiphoton process were also discussed.
Introduction Recent progress in optical microscopies has enabled direct measurements of single molecules, molecular assembles, and single particles.1-6 The application of these techniques has been providing detailed information on the nature of these systems in the individual environments. The combination of femtosecond lasers with microscopes has been utilized to improve the time resolution so as to reveal the dynamics taking place in smallsized areas. The temporal resolution under the microscope, however, is mainly limited by the dispersion due to the objective lens and typically stays in ca. 100 fs, in the case that the Ti: sapphire laser around 800 nm is employed as a light source. In view of the dispersion, it is worth noting that refractive indices of materials are usually less dependent on the wavelength in the near-infrared (NIR) region of >1 µm. Hence, it is expected that the application of the NIR laser pulse to the microscopy leads to higher resolution in time-resolved measurements. In addition to the improvement of the temporal resolution, the ultrafast NIR laser light has several advantages in the appication: (a) the low probability of light scattering can attain deeper penetration depth leading to the improved imaging of opaque materials such as biological tissues,7-12 (b) the NIR light gives less damage to the specimen, and (c) the short pulse duration easily induces higher-order multiphoton processes13-18 leading to higher spatial resolution along the optical and lateral axes. Actually, the two-photon absorption process under the optical microscope has been used as a new method to improve * E-mail:
[email protected]. Phone: +81-6-6850-6241. Fax: +81-6-6850-6244. † Graduate School of Engineering Science and Research Center for Materials Science at Extreme Conditions. ‡ Graduate School of Engineering.
10.1021/jp0561165 CCC: $33.50
the spatial resolution, because the two-photon absorption allows excitation only at the focusing point. This selectivity has been applied to improve imaging,19 microfabrication,20 optical data storage,21 stereolithography,22 and so on. Since higher-order multiphoton process needs tight confinement of photons, threeand four-photon processes may lead to higher spatial resolution than that attained by the two-photon process. Actually, it was reported that the three-photon imaging by 800 nm laser light improved the spatial resolution.23 In the following, we will present a setup of the femtosecond NIR laser microscope and its application to the detection of three- and four- photon fluorescence of organic microcrystals. Experimental Section Details of the home-built cavity-dumped Kerr-lens modelocked chromium:forsterite (Cr4+:Mg2SiO4, Cr:F) laser were described elsewhere.24,25 Briefly, the cavity was constructed by six mirrors, 19 mm Cr:F crystal as the laser medium, SF6 Brewster prism pair for compensation of the group velocity dispersion, and a Bragg cell for cavity-dumping, which was introduced to achieve high output, low repetition rate, and stability of the laser system. The Cr:F crystal was cooled to -10 °C and pumped by a CW diode-pumped Nd:YVO4 laser (Millennia IR, Spectra Physics) with output of 7 W at 1064 nm. The output pulse energy of the Cr:F laser was 12 nJ at the dumping rate of 100 kHz. Perylene (Aldrich, sublimation) and anthracene (Aldrich, zone-refined) were purified by recrystallization from ethanol. Pyrene (Wako) was chromatographed on silica gel, followed by recrystallization from ethanol. Microcrystals of perylene, anthracene, and pyrene were obtained by recrystallization from © 2006 American Chemical Society
1092 J. Phys. Chem. B, Vol. 110, No. 3, 2006
Letters
Figure 2. Images of the fluorescence of (a) perylene, (b) anthracene, and (c) pyrene microcrystals irradiated by the NIR Cr:F laser under the microscope. Scale bar: 5 µm. (d) Emission spectra of the microcrystals of anthracene (dotted line), pyrene (broken line), and perylene (smooth line) observed in the same condition. (e) The dependence of the fluorescence intensities of the pyrene (closed circle), anthracene (open circle), and perylene (open square) microcrystals on the incident NIR Cr:F laser intensity. The solid lines are results analyzed by the least-squares method. The slopes were 2.8, 3.9, and 4.3 for perylene, anthracene, and pyrene, respectively.
Figure 1. (a) Block diagram of the femtosecond near-infrared laser microscope system: PP, prism pair; λ/2, halfwave plate; OM, optical microscope; SM, fiber-coupled spectrometer; APD, avalanche photodiode; and LIA, lock-in amplifier. (b) Spectrum of Cr:F laser output. (c) Interferometric SHG autocorrelation trace and envelope curve calculated assuming a chirp-free Gaussian pulse with 35 fs fwhm.
hexane (Wako, infinity pure grade) solution. All the measurements were performed at (22 ( 1) °C. Results and Discussion Figure 1a shows a schematic block diagram of the microscope. The spectrum of the laser light covers the 1.2-1.35 µm wavelength region with a fwhm of 77 nm as shown in Figure 1b. The output of the Cr:F laser was guided into an optical microscope (IX 71, Olympus) after passing through a prism pair for the optimal compensation of the pulse duration at the sample position. Optical alignment of the Michelson interferometer was introduced for the time-resolved measurements, while this interferometer was removed when excitation intensity dependence of the multiphoton fluorescence was measured. The optical delay line (nanomover, Melles Griot) with a minimum step of 10 nm provides time-resolved measurement of every 66.7 as (attosecond) interval. A dichroic mirror (800DCSX, Chroma Technology) was introduced into the optical microscope for the selective reflection of the NIR laser pulse into an objective (MPlan 100× IR, Olympus, NA ) 0.95) and the transmittance of visible emission to the photodetector. The multiphoton fluorescence was collected by the same objective and guided into an avalanche photodiode (C5460-01, Hamamatsu). A lock-in amplifier (model 5210, EG&G Instruments) was introduced for the signal detection, which was sent to a PC for further analysis. A fiber-coupled spectrometer (SD2000, Ocean Optics) was also employed for the spectral measurements. The
multiphoton fluorescence images were measured by a CCD camera (HCC-600, Flovel). Figure 1c shows the interferometric second-order harmonic (SHG) autocorrelation trace at the sample position of the microscope after passing through the objective. The thickness of the BBO crystal was 4 mm, and the output energy for the present measurement was ca. 1 nJ/pulse. The ratio of the maximum intensity to the background was 8:1, and a symmetrical shape with respect to the time origin was obtained, indicating that a nearly ideal condition was preserved even with the dispersion of the objective. The solid line for the envelope in Figure 1c is the curve calculated on the assumption that the pulse shape and fwhm of the pulse width are Gaussian and 35 fs, respectively. The value of the time-bandwidth product was 1.2, indicating that the pulse duration under the microscope was kept almost transform-limited. This result reveals that the dispersion of ultrafast laser pulses could be minimized in the present NIR region. Figure 2a-c, respectively, show the emission images of perylene, anthracene, and pyrene microcrystals, excited with Cr:F laser (1.7 nJ/pulse) under the microscope with an optical condition similar to that employed in Figure 1c. The center of each sample irradiated with an NIR pulse shows a bright spot with a diameter of
