Abstract
Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. Thus induced two-photon absorption and the accompanied multi-photon absorption/ionization not only cause photo-bleaching but also cell damage in the vicinity of the focal point. In this paper, we study the wavelength dependent cell damage induced by high intensity femtosecond near infrared lasers. The study was performed with a Ti:sapphire laser and a Cr:forsterite laser. With a longer output wavelength from a Cr:forsterite laser, multi-photon absorption and auto-fluorescence were found to be significantly suppressed and the destructive plasma formation was found to be greatly reduced. Sustained multi-photon spectra can be observed in most plant specimens with a tightly focused Cr:forsterite laser beam under long term irradiation with more than 100 mW laser average power. In contrast, multi-photon absorption induced destructive plasma formation were frequently observed with a tightly focused Ti:sapphire laser beam within seconds with more than 10 mW laser average power.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Agarie, S., W. Agata, H. Uchida, F. Kubota and P.B. Kaufman. Function of silica bodies in the epidermal system of rice (Oryza sativa L.): Testing the window hypothesis. J. Exp. Bot. 47 655, 1996.
Anderson, R.R., S. Zaynoun and J.A. Parish. Cumulative effects of repeated subthreshold doses of ultraviolet radiation. J. Invest. Dermatol. 77 13, 1981.
Barad, Y., H. Eisenberg, M. Horowitz and Y. Silberberg. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70 922, 1997.
Cheng, P.C., F.-J. Kao, C.-K. Sun, B.-L. Lin, T.-M. Liu, Y.-S. Wang, M.-K. Huang, Y.-M. Wang, J.-C. Chen and I. Johnson. Multi-photon excited fluorescence spectra of common bio-probes. Scanning 22 187, 2000.
Cheng, P.C., B.-L. Lin, F.-J. Kao, M. Gu, M.-G. Xu, X Gan, M.-K. Huang and Y.-S. Wang. Multiphoton fluorescence microscopy-the response of plant cells to high intensity illumination. Micron 32, 661, 2001.
Cheng, P.C., S.J. Pan, A. Shih, K.-S. Kim, W.S. Liou and M.S. Park. Highly efficient upconverters for multiphoton fluorescence microscopy. J. Microsc. 189 199, 1998.
Chu, S.-W., I.-H. Chen, T.-M. Liu, P.C. Cheng, C.-K. Sun and B.-L. Lin. Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser. Opt. Lett. 26 1909, 2001.
Denk, W., J.H. Strickler and W.W. Webb. Two-photon laser scanning fluorescence microscopy. Science 248 73, 1990.
Iwamoto, M., K.H. Norris and S. Kimura. Rapid prediction of chemical compositions for wheat, soybean, pork and fresh potatoes by near infrared spectrophotometric analysis. Nippon Shokuhin Kogyo Gakkaishi 28 85, 1981.
Kennedy, S.M. and F.E. Lytle. p-Bis(io-methylstyryl)benzene as a power squared sensor for two-photon absorption measurment between 537 and 694 nm. Anal. Chem. 58 2643, 1986.
König, K., H. Liang, M.W. Berns and B.J. Tromberg. Cell damage by near-IR microbeams. Nature 377 20, 1995.
König, K., P.T.C. So, W.W. Mantulin and E. Gratton. Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. Opt. Lett. 22 135, 1997.
Liu, T.-M., S.-P. Tai and C.-K. Sun. Intracavity frequency-doubled femtosecond Cr4+:forsterite laser. Appl. Opt. 40 1957, 2001a.
Liu, T.-M., S.-H. Chu, C.-K. Sun, B.-L. Lin, P.C. Cheng and I. Johnson. Multiphoton confocal microscopy by using a femtosecond Cr:forsterite laser. Scanning 23 in press, 2001b.
Seas, A., V. Petričević and R.R. Alfano. Generation of sub-100-fs pulses from a CW mode-locked chromium-doped forsterite laser. Opt. Lett. 17 937, 1992.
Seas, A., V. Petričević and R.R Alfano. Self-mode-locked chromium-doped forsterite laser generates 50-fs pulses. Opt. Lett. 18 891, 1993.
Sun, C.-K., S.-W. Chu, S.P. Tai, S. Keller, U.K. Mishra and S.P. DenBaars. Scanning second-harmonic/ third-harmonic generation microscopy of gallium nitride. Appl. Phys. Lett. 77 2331, 2000.
Slobodchikov, E., J. Ma, V. Kamalov, K. Tominaga and K. Yoshihara. Cavity-dumped femtosecond Kerr-lens mode locking in a chromium-doped forsterite laser. Opt. Lett. 21 354, 1996.
Squier, J.A., M. Müller, G.J. Brakenho. and K.R. Wilson. Third harmonic generation microscopy. Opt. Exp. 3 315, 1998.
Stelzer, E.H.K., S. Hell, S. Lindek, R. Stricker, R. Pick, C. Storz, G. Ritter and N. Salmon. Nonlinear absorption extends confocal fluorescence microscopy into the ultra-violet regime and confines the illumination volume. Opt. Commun. 104 233, 1994.
Zhang, Z., K. Torizuka, T. Itatani, K. Kobayashi, T. Sugaya and T. Nakagawa. Femtosecond Cr:forsterite laser with mode locking initiated by a quantum-well saturable absorber. IEEE. J. Quantum Electron. 33 1975, 1997.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chen, IH., Chu, SW., Sun, CK. et al. Wavelength dependent damage in biological multi-photon confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources. Optical and Quantum Electronics 34, 1251–1266 (2002). https://doi.org/10.1023/A:1021303426482
Issue Date:
DOI: https://doi.org/10.1023/A:1021303426482