We present multiphoton microendoscopy with a rotational probe and a 1 microm fiber-based femtosecond laser. The rotational probe is based on a double-clad photonic crystal fiber, a gradient index lens, a microprism, and a rotational microelectronicmechanical system (MEMS) motor. The MEMS motor has a diameter of 2.2 mm and can provide 360 degrees full-view rotation. The fiber laser provides ultrashort pulses with a central wavelength at 1.034 microm and a repetition rate of 50 MHz. Second-harmonic-generation images of rat-tail tendon and fish scale are demonstrated with the rotational probe-based multiphoton system

Chen, Zhongping

Liu, Gangjun

Su, Jianping

Tomov, Ivan V

Tromberg, Bruce J

Xie, Tuqiang

Yu, Lingfeng

Zhang, Jun

eScholarship - University of California

UC IrvineUC Irvine Previously Published WorksTitleRotational multiphoton endoscopy with a 1 microm fiber laser system.Permalinkhttps://escholarship.org/uc/item/64f0f9t2JournalOptics Letters, 34(15)ISSN0146-9592AuthorsLiu, GangjunXie, TuqiangTomov, Ivan Vet al.Publication Date2009-08-01DOI10.1364/ol.34.002249Copyright InformationThis work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaRotational multiphoton endoscopy with a 1 μm fiber lasersystemGangjun Liu1, Tuqiang Xie1, Ivan V. Tomov1, Jianping Su2, Lingfeng Yu2, Jun Zhang1,Bruce J. Tromberg1, and Zhongping Chen1,2,*1 Beckman Laser Institute, University of California, Irvine, Irvine, California 92612, USA2 Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92617,USAAbstractWe present multiphoton microendoscopy with a rotational probe and a 1 μm fiber-basedfemtosecond laser. The rotational probe is based on a double-clad photonic crystal fiber, a gradientindex lens, a microprism, and a rotational microelectronicmechanical system (MEMS) motor. TheMEMS motor has a diameter of 2.2 mm and can provide 360° full-view rotation. The fiber laserprovides ultrashort pulses with a central wavelength at 1.034 μm and a repetition rate of 50 MHz.Second-harmonic-generation images of rat-tail tendon and fish scale are demonstrated with therotational probe-based multiphoton system.Multiphoton optical microscopy (MPM) is a powerful tool in the imaging sciences.Compared with their linear counterpart, MPM allows deeper penetration into thick tissuesowing to near-IR excitation wavelength used [1]. Because the excitation is confined to thefocal region in MPM, out-of-focus photobleaching and photodamage is greatly reduced [1].Since the introduction of MPM, these tools have been widely used in the fields of biologicaland medical sciences.Commercially available femtosecond Ti:sapphire lasers are the most commonly used lasersources for MPM. Ti:sapphire lasers are broadly tunable from 700 to 1000 nm. A longerwavelength source beyond 1 μm is also of interest. At longer excitation wavelengths,penetration depth is greater owing to reduced scattering coefficient at a longer excitationwavelength [2]. When shifting the excitation wavelength from 800 to 1000 nm and keepingthe other factors the same, imaging depth will be increased greatly for tissues such as skin,muscle, and tumor [2]. A 1.3 μm source may improve the imaging depth twofold ascompared with a conventional 800 nm source [3]. Fiber lasers may operate at 1.0, 1.3, and1.5 μm [2]. With the light confined in fiber, fiber-based sources are more compact and notsusceptible to misalignment. Fiber-based sources with fiber output allow direct couplingwith a fiber-based system.Miniaturization of the nonlinear microscopy is of great interest for imaging tissues in vivoand ex vivo. The challenges for the miniaturization are efficient excitation pulse deliverywith minimal pulse distortion, efficient multiphoton signal collection, and miniaturescanning mechanism. Various fibers, including single-mode fiber, hollow core fiber, fiberbundle, and double-clad photonic crystal fiber (DCPCF), have been investigated for MPMendoscopy applications [4–8]. Of these, the DCPCF is unique because it can support single-mode propagation of the excitation wavelength through a relatively large area core while*Corresponding author: z2chen@uci.edu.NIH Public AccessAuthor ManuscriptOpt Lett. Author manuscript; available in PMC 2011 March 16.Published in final edited form as:Opt Lett. 2009 August 1; 34(15): 2249–2251.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsimultaneously supporting the multimode propagation of the multiphoton signal through thehigh-NA inner clad [6,8]. The relatively large-area core can significantly reduce the powerdensity in the core and, therefore, minimize the nonlinear effects. The clad has a largediameter with a high NA, and high signal collection efficiency can be achieved [6,8]. For thescanning mechanism, two-axis micro-electronicmechanical system (MEMS) scanningmirror, piezoelectric, and electromagnetic actuators have been proposed to scan the laserbeam [4,8–11]. The piezoelectric and electromagnetic actuators provide a forward spiral orLissajous scan pattern, and the MEMS mirrors provide two axes scanning from a sideviewdirection.For biomedical imaging applications such as the in-travascular and gastrointestinal tract,sideview rotational imaging is preferred owing to their hollow feature. In this Letter, wepropose MPM that uses a rotational microprobe and a 1 μm fiber laser. The rotational probeconsists of a DCPCF, a gradient index (GRIN) lens, a microprism, and a rotational MEMSmotor. The fiber laser produces ultrashort femtosecond pulses with a central wavelength at1.034 μm, a spectral width of 13 nm, and a repetition rate of 50 MHz. Two-dimensionalbeam scanning is realized by the linear transversal scanning provided by a linear stage androtational scanning by an MEMS motor.Our system setup is shown in Fig. 1. A home-built, mode-locked, self-starting, stretched-pulse, Yb-doped fiber laser generates ultrashort pulses with the central wavelength at 1.034μm and a repetition rate of 50 MHz [12]. The mode-locked pulses from the fiber oscillatorare temporally stretched in a 10 m single-mode fiber and amplified in yttrium-doped fiberpumped by two 980 nm diode lasers. A polarized pulse with a maximum output power of270 mW is obtained after dechirping by an external grating pair. Because most of the partsare fiber based in this laser, the entire laser source occupies a dimension of 30 cm × 45 cm ×12 cm, and it can be compacted into a much smaller space. Laser beam after the grating pairis coupled into a DCPCF (Crystal Fiber A/S, DC-165-16-PASSIVE) by a microscopeobjective. The output beam pattern from the DCPCF is monitored to guarantee the coremode operation. In our current configuration, the coupling efficiency of the excitation laserbeam to the DCPCF is approximately 50%. More than 50% of the total output power goingthrough the DCPCF is in the core of the fiber. The core mode power is measured byspatially filtering the light in the clad with an iris. The laser beam through the DCPCF issent to a homemade interference autocorrelater to monitor the pulse width. By adjusting thedistance between the grating pair and monitoring the interference autocorrelation trace of theoutput pulse, we get the shortest pulse through the DCPCF. The shortest pulse width throughthe DCPCF is 290 fs after dispersion compensation. The multiphoton signal collected by theDCPCF is separated from the input via a dichroic beam-splitter and then focused on aphotomultiplier tube (PMT) after passing through a short-pass emission filter. Signal fromthe PMT is further amplified by a low-noise pre-amplifier (Stanford Research Systems, Inc.,SR560) before being sent to a personal computer for data acquisition (DAQ) and processing.For initially testing of the DCPCF and GRIN lens for the 1 μm fiber laser system, we used atwo-photon diode (Hamamatsu Photonics, G1115) as a sample and detected the two-photonsignal versus the incident power. The two-photon diode has a spectral range of 300–680 nm,and its output was amplified by a low-noise-current preamplifier (Stanford ResearchSystems, Inc., SR570). The quadratic power dependence confirms the two-photon nature ofthe signal. To further test the excitation and signal collection efficiency of our system, weused potassium dihydrogen phosphate (KDP) crystal powder as a sample and tried to findthe relationship between the excitation power and second-harmonic signal collected by theDCPCF. Figure 2 shows this relationship. The quadratic power dependence of the second-harmonic-generation (SHG) signal on the excitation power can be seen from the fitted curve.Liu et al. Page 2Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptWe have developed a sideview imaging rotational probe for optical coherent tomographyendoscopy [13]. However, to our knowledge this kind of probe has not been demonstrated inthe nonlinear microscopy field. The schematic of the mircorotational probe is illustrated inFig. 3(a). Light passed through the DCPCF is focused by a GRIN lens. The focusing beamafter the GRIN lens is reflected by a right-angle microprism glued to the shaft of themicrorotational motor. The 1 mm microprism is coated with aluminum, and it provides morethan 90% reflection efficiency. The MEMS rotational motor is 2.2 mm in diameter and 12mm in length, including the shaft. The rotational motor is driven by an external motorcontroller through a control wire. The MEMS rotational motor can reach about 40 rounds/swithout any load on the shaft. With the microprism glued to the shaft, the rotation speeddrops to 5 rounds/s. The only moving part in this configuration is the microprism glued tothe shaft of the MEMS motor. Figure 3(b) shows the photograph of the rotational MEMSmotor glued with the microprism together with a United States dime coin.Collagen is the main structural protein and fibrous component for tendon, skin, bone, andcartilage. Collagen may be highly ordered and possesses a large second-order nonlinearsusceptibility. It is known that rat-tail tendon and fish scale consist of dense collagen fibrils,and they are expected to yield a bright SHG signal. In this study, the tail was removed froma rat sacrificed from another experiment and was frozen for hours before our experiment.The rat-tail tendon was removed from the thawed rat tail and imaged. Scales was pluckedfrom live fish and kept at room temperature for hours before experiment. Figures 4(a) and4(b) show the SHG images of the rat-tail tendon and the fish scale. The scale bar is 25 μm.X-direction (horizontal direction in the picture) scanning is realized by the rotational probe.Y-direction scanning (vertical direction in the picture) is realized by a translation stage.Rotation speed of the MEMS motor is set at 1 round/s to enhance the contrast. Theexcitation power delivered on the sample is 50 mW (through the core of the fiber based on50% of the total output power in the core). When processing the data, background noise issubtracted first. Background noise is determined by the DAQ signal without the sample. Thebackground-noise-subtracted signal is expressed in eigth-bit pseudocolor according to thesignal value. In the results shown here, we demonstrate only a 100 μm imaging range in therotation direction. The coated microprism could achieve a 360° full-circle view so the probecould realize 360° rotational imaging. The packaged probe could also be designed as aneedle type for deep-tissue imaging.In conclusion, we demonstrated a design of a rotational MPM probe and used a 1 μm fiberlaser as a laser source. The probe is based on a DCPCF, a GRIN lens, a microprism, and anMEMS motor. We experimentally verified the feasibility of the DCPCF fiber as signaldelivery and multiphoton signal collection media for an MPM system with 1 μm excitationwavelength. SHG images were demonstrated with the rotational probe and a fiber-laser-based MPM system. The probe can achieve a 360° full-circle view and could realize a deep-tissue, large-imaging range if packaged in a needle form.AcknowledgmentsThis work was supported by the National Institutes of Health (NIH) (EB-00293, NCI-91717, RR-01192), NationalScience Foundation (NSF) (BES-86924), Air Force Office of Scientific Research (AFOSR) (FA9550-04-0101), andthe Beckman Laser Institute Endowment.References1. Helmchen F, Denk W. Nat Med. 2005; 2:932.2. Tromberg, BJ. Handbook of Biomedical Nonlinear Optical Microscopy. Masters, BR.; So, PTC.,editors. Oxford: U. Press; 2008. p. 707-715.Liu et al. Page 3Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript3. Balu M, Baldacchini T, Carter J, Krasieva TB, Zadoyan R, Tromberg BJ. J Biomed Opt. 2009;14:010508–1. [PubMed: 19256688]4. Jung JC, Schnitzer MJ. Opt Lett. 2003; 28:902. [PubMed: 12816240]5. Flusberg BA, Cocker ED, Piyawattanametha W, Jung JC, Cheung ELM, Schnitzer MJ. Nat Med.2005; 2:941.6. Myaing MT, Ye JY, Norris TB, Thomas T, Baker JR Jr, Wadsworth WJ, Bouwmans G, Knight JC,St Russell PJ. Opt Lett. 2003; 28:1224. [PubMed: 12885028]7. Göbel W, Kerr JND, Nimmerjahn AL, Helmchen F. Opt Lett. 2004; 29:2521. [PubMed: 15584281]8. Fu L, Gan X, Gu M. Opt Express. 2005; 13:5528. [PubMed: 19498549]9. Jung W, Tang S, McCormic DT, Xie T, Ahn Y, Su J, Tomov IV, Krasieva TB, Tromberg BJ, ChenZ. Opt Lett. 2008; 33:1324. [PubMed: 18552946]10. Myaing MT, MacDonald DJ, Li X. Opt Lett. 2006; 31:1076. [PubMed: 16625908]11. Engelbrecht1 CJ, Johnston RS, Seibel EJ, Helmchen F. Opt Express. 2008; 16:5556. [PubMed:18542658]12. Lim H, Ilday FÖ, Wise FW. Opt Lett. 2003; 28:660. [PubMed: 12703933]13. Su J, Zhang J, Yu L, Chen Z. Opt Express. 2007; 15:10390. [PubMed: 19547391]Liu et al. Page 4Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 1.Diagram of the multiphoton imaging system. A, fiber laser oscillator; B, fiber amplifier; C,grating pair compressor; D, the microprobe, data acquisition, and processing system. WDM,wavelength-division multiplexer; PCF, photonic crystal fiber; HWP, half-wave plate; PMT,photo-multiplier tube; AMP, amplifier.Liu et al. Page 5Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 2.Double-logarithmic plot of the SHG intensity from KDP crystal power as a function ofincident power.Liu et al. Page 6Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 3.(Color online) Diagram of the microrotational probe. The probe is based on a DCPCF, aGRIN lens, a microprism, and a rotational MEMS motor. (b) Photograph of the microprismglued to a MEMS motor together with a United States dime coin.Liu et al. Page 7Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFig. 4.(Color online) SHG images of (a) a rat tail tendon and (b) a fish scale. Scale bar is 25 μm.Liu et al. Page 8Opt Lett. Author manuscript; available in PMC 2011 March 16.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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Rotational multiphoton endoscopy with a 1 microm fiber laser system.

Rotational multiphoton endoscopy with a 1 microm fiber laser system.

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