A 0.4-mm-diameter probe for nonlinear optical imaging Hongchun Bao and Min Gu* Centre for Micro-Photonics, Faculty of Engineering & Industrial Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia mgu@swin.edu.au Abstract: A miniaturized probe that possesses a diameter of 0.4 mm is developed for two-photon-excited fluorescence imaging. The miniaturized probe was manufactured by the collapse of air holes and the formation of a lens on the tip of a double-clad photonic crystal fiber (DCPCF) using electric arc discharging from a conventional fusion splicer. As a result, a femtosecond pulsed laser beam delivered by the DCPCF can be directly focused on a sample for two-photon fluorescence imaging. The numerical aperture of the lensed DCPCF is 0.12. The corresponding focal spot size is 6 ��m, which is close to the diffraction limit. This 0.4-mm-diamter probe can provide clear two-photon-excited fluorescence images of 10-��m-diameter fluorescent microspheres. ��2009 Optical Society of America OCIS codes: (110.2350) Fiber optical imaging (180.4315) Nonlinear microscopy (110.3080) Infrared imaging (110.6880) Three-dimensional image acquisition (170.2150) Endoscopic imaging. References and links 1. W. Denk, J. H. Strickler, and W. W. Webb, ���Two-photon laser scanning fluorescence microscopy,��� Science 248(4951), 73���76 (1990). 2. H. Bao, J. Allen, R. Pattie, R. Vance, and M. Gu, ���Fast handheld two-photon fluorescence microendoscope with a 475 microm x 475 microm field of view for in vivo imaging,��� Opt. Lett. 33(12), 1333���1335 (2008). 3. B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P. Anderson, and M. J. Schnitzer, ���In vivo brain imaging using a portable 3.9 gram two-photon fluorescence microendoscope,��� Opt. Lett. 30(17), 2272���2274 (2005). 4. K. K��nig, A. Ehlers, I. Riemann, S. Schenkl, R. B��ckle, and M. Kaatz, ���Clinical two-photon microendoscopy,��� Microsc. Res. Tech. 70(5), 398���402 (2007). 5. T. P. Thomas, J. Y. Ye, Y. C. Chang, A. Kotlyar, Z. Cao, I. J. Majoros, T. B. Norris, and J. R. Baker, Jr., ���Investigation of tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probe,��� J. Biomed. Opt. 13(1), 014024 (2008). 6. D. Bird, and M. Gu, ���Compact two-photon fluorescence microscope based on a single-mode fiber coupler,��� Opt. Lett. 27(12), 1031���1033 (2002). 7. D. Bird, and M. Gu, ���Two-photon fluorescence endoscopy with a micro-optic scanning head,��� Opt. Lett. 28(17), 1552���1554 (2003). 8. D. Bird, and M. Gu, ���Fibre-optic two-photon scanning fluorescence microscopy,��� J. Microsc. 208(1), 35���48 (2002). 9. L. Fu, X. Gan, and M. Gu, ���Use of a single-mode fiber coupler for second-harmonic-generation microscopy,��� Opt. Lett. 30(4), 385���387 (2005). 10. F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk, ���A miniature head-mounted two-photon microscope. high- resolution brain imaging in freely moving animals,��� Neuron 31(6), 903���912 (2001). 11. M. T. Myaing, D. J. MacDonald, and X. Li, ���Fiber-optic scanning two-photon fluorescence endoscope,��� Opt. Lett. 31(8), 1076���1078 (2006). 12. L. Fu, X. Gan, and M. Gu, ���Nonlinear optical microscopy based on double-clad photonic crystal fibers,��� Opt. Express 13(14), 5528 (2005). 13. L. Fu, A. Jain, H. Xie, C. Cranfield, and M. Gu, ���Nonlinear optical endoscopy based on a double-clad photonic crystal fiber and a MEMS mirror,��� Opt. Express 14(3), 1027���1032 (2006). 14. L. Fu, and M. Gu, ���Double-clad photonic crystal fiber coupler for compact nonlinear optical microscopy imaging,��� Opt. Lett. 31(10), 1471���1473 (2006). 15. L. Fu, A. Jain, C. Cranfield, H. Xie, and M. Gu, ���Three-dimensional nonlinear optical endoscopy,��� J. Biomed. Opt. 12(4), 040501 (2007). #109016 - $15.00 USD Received 20 Mar 2009 revised 27 May 2009 accepted 27 May 2009 published 1 Jun 2009 (C) 2009 OSA 8 June 2009 / Vol. 17, No. 12 / OPTICS EXPRESS 10098

16. M. T. Myaing, J. Y. Ye, T. B. Norris, T. Thomas, J. R. Baker, Jr., W. J. Wadsworth, G. Bouwmans, J. C. Knight, and P. S. Russell, ���Enhanced two-photon biosensing with double-clad photonic crystal fibers,��� Opt. Lett. 28(14), 1224���1226 (2003). 17. L. Fu, and M. Gu, ���Fibre-optic nonlinear optical microscopy and endoscopy,��� J. Microsc. 226(3), 195���206 (2007). 18. C. L. Hoy, N. J. Durr, P. Chen, W. Piyawattanametha, H. Ra, O. Solgaard, and A. Ben-Yakar, ���Miniaturized probe for femtosecond laser microsurgery and two-photon imaging,��� Opt. Express 16(13), 9996���10005 (2008). 19. D. Yelin, I. Rizvi, W. M. White, J. T. Motz, T. Hasan, B. E. Bouma, and G. J. Tearney, ���Three-dimensional miniature endoscopy,��� Nature 443(7113), 765 (2006). 20. T. P. Thomas, J. Y. Ye, C. Yang, M. Myaing, I. J. Majoros, A. Kotlyar, Z. Cao, T. B. Norris, and R. James, ���Baker Jr, ���Tissue distribution and real-time fluorescence measurement of a tumor-targeted nanodevice by a two photon optical fiber fluorescence probe,��� Proc. SPIE 6095, 60950Q (2006). 21. C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F. Helmchen, ���Ultra-compact fiber-optic two-photon microscope for functional fluorescence imaging in vivo,��� Opt. Express 16(8), 5556���5564 (2008). 22. G. J. Kong, J. Kim, H. Y. Choi, J. E. Im, B. H. Park, U. C. Paek, and B. H. Lee, ���Lensed photonic crystal fiber obtained by use of an arc discharge,��� Opt. Lett. 31(7), 894���896 (2006). 23. H. Bao, and M. Gu, ���Reduction of self-phase modulation in double-clad photonic crystal fiber for nonlinear optical endoscopy,��� Opt. Lett. 34(2), 148���150 (2009). 24. N. Mihajlovic, G.W. ���t Hooft, B.H.W. Hendriks, W.C.J. Bierhoff, C.A. Hezemans, R. Harbers, A.L. Braun, J.J.L. Horikx, and A.E. Desjardins, ���Electromagnetically-Controlled Fiber-Scanning Confocal Microscope,��� OSA Optics & Photonics Congress, NWC5, (2009). 1. Introduction Nonlinear optical microscopy based on multi-photon-excited fluorescence uses a near infrared laser beam for imaging, which could identify cell structures of tissue deep beneath the surface [1���3]. However, despite of its usefulness, nonlinear microscopy imaging using a standard laboratory microscopy with inflexible free-space light delivery limits its applications especially for clinical or animal studies. A flexible fiber-optical probe is versatile to deliver light into tight space where free-space delivery is difficult [4,5]. So far, three different types of fiber have been adopted for multi-photon-excited fluorescence imaging. Single-mode fiber was first used for this purpose [6���10] but such a system exhibits a low signal level because the near infrared laser beam and the visible fluorescence signal cannot efficiently propagate through the core of the fiber [8]. This issue has been overcome by using a double-clad fiber [2,11]. In fact, the signal level can be further increased when a double-clad photonic crystal fiber (DCPCF) that has a large core is applied [12���17]. In all these micro-probe designs, coupling optics has been used to deliver the excitation beam and collect fluorescence, which eventually leads to a probe size over 1.5 mm [2,10,18]. Reducing the size of probes is essential for minimizing invasion during medical procedures and reducing the risk of complications as well as costs and recovery times [19��� 21]. In this paper, we demonstrate a small probe with a diameter of 0.4 mm, which is only one third of the size of a normal micro-probe [15]. The 0.4-mm-diameter probe is developed by the formation of a semi-sphere lens on the tip of a DCPCF which is used for delivering a femtosecond pulsed laser beam to samples and collecting the two-photon fluorescence signal for imaging. The 0.4-mm-probe can produce a diffraction-limited focal spot with a full width at half-maximum (FWHM) of 6 ��m and thus provide clear images of 10 ��m fluorescent microspheres. 2. Two-photon-excited fluorescence imaging system Figure 1 is the schematic setup for two-photon-excited fluorescence imaging using the 0.4- mm-diameter optical probe. A DCPCF is used to deliver 80 MHz repetition rate pulses of 100 fs from a Ti:Sapphire laser to a sample to excite two-photon fluorescence signal. The excitation laser beam has a center wavelength of 800 nm with a FWHM bandwidth of 12 nm. In order to keep a short pulse width, a prechirp unit ��� a grating pair is used to implement negative frequency chirping to the pulses from the Ti:Sapphire laser [2], so that the chromatic dispersion of the DCPCF is compensated. The schematic structure of the DCPCF is displayed in the low left corner of Fig. 1. The solid core of the DCPCF, which is in the center of the #109016 - $15.00 USD Received 20 Mar 2009 revised 27 May 2009 accepted 27 May 2009 published 1 Jun 2009 (C) 2009 OSA 8 June 2009 / Vol. 17, No. 12 / OPTICS EXPRESS 10099