We report the investigation and implementation of a compact second harmonic generation microscope that uses a single-mode fiber coupler and a double-clad photonic crystal fiber. Second harmonic polarization anisotropy through the fiber-optic microscope systems is quantitatively measured with KTP microcrystals, fish scale and rat tail tendon. It is demonstrated that the polarized second harmonic signals can be excited and collected through the single-mode fiber coupler to analyze the molecular orientations of structural proteins. It has been discovered that a double-clad photonic crystal fiber can preserve the linear polarization in the core, although a depolarization effect is observed in the inner cladding region. The feasibility of polarization anisotropy measurements in fiber-optic second harmonic generation microscopy will benefit the in vivo study of collagen-related diseases with a compact imaging probe.
©2008 Optical Society of America
Second harmonic generation (SHG) microscopy is a noninvasive optical imaging modality for three-dimensional high-resolution visualization of endogenous arrays of collagen, microtubules and muscle myosin in wide variety of cells and tissues [1–3]. The coherent process in SHG enables the polarization dependence of harmonic light that provides information about molecular organization and nonlinear susceptibilities not available from fluorescence light with random phase [4–7]. Different from conventional polarization microscopy examining the linear birefringence of samples, SHG microscopy can obtain the absolute orientation of molecules by use of arbitrary combinations of fundamental and harmonic polarization states. Due to the polarization anisotropy nature, SHG microscopy has been combined with multi-photon fluorescence microscopy [8–9] and optical coherent tomography [10–12] to enhance the imaging contrast for morphology identification in biological tissue.
Historically, SHG polarization anisotropy is studied with bulk optical systems that permit precise control of polarization states of light, however, preclude in vivo imaging for internal organs and behaving animals. Integration of optical fibers [13–18] into SHG microscopy can offer a mechanical flexibility for system arrangement but pose problems of polarization anisotropy measurements. The difficulty in achieving SHG polarization anisotropy through fiber-optic microscope systems is to preserve linear polarization of both ultrashort pulses and SHG signals over the wide wavelength range. Recently, the imaging capability of SHG microscopy using a single-mode fiber (SMF) coupler  and a double-clad photonic crystal fiber (PCF)  have been demonstrated, respectively. In addition, pilot studies have shown that photonic crystal fibers exhibit appreciable polarization properties [21–24]. However, there has not been an investigation of how these fiber-optic SHG microscopes can implement SHG polarization anisotropy and the performance of different types of fibers in the measurement. Here we present the polarization characteristics of fiber-optic SHG microscopy using an SMF coupler and a double-clad PCF, particularly the polarization anisotropy measurements with KTP microcrystals, fish scale, and rat tail tendon.
2. Experimental arrangements
A schematic diagram of the experiment setup for measuring the polarization anisotropy of the fiber-optic SHG microscope is shown in Fig. 1. In the SHG microscope using an SMF coupler (Newport, F-CPL-S12785) shown in Fig. 1(a), a pulsed beam generated from a Ti:Sapphire laser (Spectra Physics, Mai Tai) at wavelength 800 nm with a repetition rate of 80 MHz and a pulse width of approximately 80 fs is delivered through the excitation arm to the sample arm, and the SHG signal is collected via the sample arm and the signal arm into a photomultiplier tube (PMT). The SMF coupler we used has a core/cladding ratio of approximately 5/125, numerical aperture (NA) 0.16, and an operation wavelength of 780 nm. The coupler is analogous to a dichroic mirror (DCM) in a conventional microscope and the fiber tips act as pinholes to reduce the multiple scattering of signals . When a double-clad PCF (Crystal Fiber A/S) is adopted to enhance the signal level of the SHG microscope, the SMF coupler is replaced by a double-clad PCF and a DCM, whose reflectance is independent on the polarization state of the light (Fig. 1(b)). Having a core diameter of 20 µm, an inner cladding with a diameter of 165 µm, and an NA of 0.6 at wavelength 800 nm, the double-clad PCF offers simultaneous single-mode delivery in the core at a near infrared wavelength and multimode collection through the inner cladding for visible SHG signals . In both cases, the objectives in the measurements have low NA, therefore have no depolarization effect on the polarized light. The fibers are in natural status and arranged to avoid strain and stress. Experimental results do not show the sensitivity with respect to fiber bending and stress.
To investigate the evolution of input polarization states, an arbitrary linear polarization direction is made by the rotation of the λ/4 plate and the Glan Thompson polarizer (GTP, Newport: 10GT04). For a given incident polarization angle θi at the input port of fibers, the maximum (Imax) and minimum (Imin) intensity of the output beam are measured through an analyzer (GTP, Newport: 10GT04) to determine the degree of polarization as γ=(Imax-Imin)/(Imax+Imin). The SHG polarization anisotropy is measured by obtaining images through rotations of the analyzer before the PMT while maintaining the excitation polarization after the fiber. In experiments, the initial rotation angle of the analyzer, corresponding to the maximum SHG intensity, is parallel to the excitation polarization. A 400/9 nm bandpass filter (BF) is placed before the PMT to ensure that only the SHG signal is detected. The sample is scanned two-dimensionally by a scanning stage (Physik Instrumente).
3. SHG polarization anisotropy using an SMF coupler
To study the ability of the fiber-optic SHG microscope for polarization anisotropy measurements, it is important to understand the polarization characteristics of the SMF coupler under various illumination conditions. The prior results show that the linear polarization states of pulsed and continuous laser beams over a range from near infrared to visible wavelengths can be maintained in the conventional SMF coupler due to the birefringence effect . Furthermore, polarization preservations appear at an angular interval of approximately 90° of the incident polarization angle with the respect to the transverse axes of the fiber. It implies that the SMF coupler enables the delivery of a linearly polarized excitation beam and the propagation of the SHG signal. To apply this knowledge to fiber-optic SHG microscopy imaging, a standard nonlinear optical crystal, KTP microcrystals (Shandong University, China), is used as a sample to give well-polarized SHG emission under the linear excitation polarization.
We first quantitatively analyze and compare the polarization anisotropy of the KTP microcrystals in a commercial nonlinear laser scanning microscope (Olympus, Fluoview 300, epi-detection) and that in an SMF coupler-based SHG microscope. When the laser excitation polarization is fixed, SHG signals are expected to have parallel polarization with the laser and therefore should yield a cos2 θ pattern by rotating the analyzer before the PMT. Successive SHG images of the microcrystals are recorded from both microscope systems when the laser with linear polarization is delivered and the analyzer is rotated by 180° at a step of 10°. Figures 2(a–b) and 2(c–d) show SHG images of the KTP microcrystals at orthogonal polarization orientations of the analyzer obtained from the standard laser scanning microscope and the SMF coupler-based microscope, respectively. In both cases, the extracted SHG intensity as a function of the analyzer rotation angle is well consistent with the prediction based on a cos2 θ pattern, which can be observed from Fig. 2(e). It is demonstrated that the SHG microscope using an SMF coupler exhibits the same manner of SHG polarization anisotropy compared with that in the conventional SHG microscope with bulk optics. The deviation of the experimental data from the theoretical expectation may arise from the depolarization effects of galvanometric mirrors and the imaging objective.
The capability of the system for the polarization anisotropy measurement is further confirmed by the SHG signals obtained from a tetra fish scale (Figs. 3(a–b)). The fish scale consists of abundance of well-structured collagen fibrils, which are corresponded to SHG signals. The observation from Figs. 3(a) and 3(b) reveals that the collagen fibrils in fish scale is highly anisotropic. In particular, molecular orientation in fish scale can be quantified by the anisotropy parameter β=(Imax-Imin)/(Imax+2 Imin), where Imax and Imin are the SHG intensity with the polarization parallel and perpendicular to the incident polarization (Fig. 3(c)). Measured β values in most areas in Fig. 3(c) are greater than 0.7, which is consistent with the prior results obtained from a standard SHG microscope  and indicates a good alignment of collagen fibrils relative to the incident polarization.
4. SHG polarization anisotropy using a double-clad PCF
As mentioned in the Introduction, a new development in fiber-optic nonlinear optical microscopy occurs after the introduction of a double-clad PCF results in a signal level improvement of two orders of magnitude . Based on a double-clad PCF, a microelectromechanical system (MEMS) mirror, and a gradient index (GRIN) lens, three-dimensional nonlinear optical endoscopic imaging through tissue has become possible [25,26]. The coupling efficiency and SHG imaging through the double-clad PCF have been demonstrated elsewhere [20,25]. To investigate its polarization characteristics, the output polarization states of the laser beam at wavelength 800 nm as a function of the incident polarization angle θi are measured (Fig. 4). It is found that the maximum degree of polarization of the output laser beam is approximately 0.31 through the fiber core and the inner cladding, exhibiting an angular interval of approximately 90° (Fig. 4(a)). However, the degree of polarization of approximately 0.84 appears in the central core (Fig. 4(b)), where the ultrashort pulsed light for SHG imaging is actually propagated due to minimized dispersion.
Consequently, this result indicates that a linearly polarized excitation beam can be delivered through the fiber core for SHG anisotropy measurement. It should be noted that the double-clad PCF does not provide a degree of polarization of approximately 1, as has been demonstrated in the SMF coupler. It may be caused by the depolarization effect of the large core area and the microstructures in the inner cladding.
Under the experimental condition where the linearly polarized light at 800 nm is delivered by the fiber core, SHG polarization anisotropy measurements in microscopy using a double-clad PCF are shown in Fig. 5. The tendon is extracted axially through the tail tendon sheathing of a Sprague-Dawley rat and attached directly to the coverslip. For both fish scale and rat tail tendon, SHG images are obtained in the cases of no analyzer before PMT and orthogonal polarization orientations of the analyzer. The anisotropy parameter images in Fig. 5 show that the average β value is approximately 0.15 for the fish scale and 0.2 for the rat tail tendon, demonstrating that SHG signals from the two highly ordered samples experience depolarization through the double-clad PCF. This result implies that photonic crystal structures in the inner cladding of the fiber, which enable the enhancement of the SHG collection efficiency, however result in the significant depolarization effect over the near infrared and the visible wavelength ranges.
We have demonstrated for the first time to our knowledge SHG polarization anisotropy measurements by using an SMF coupler and a double-clad PCF. Our results show that the SMF coupler offers polarization preservations for the ultrashort pulsed excitation beam at near infrared wavelength and the visible SHG signal, and therefore enables polarization anisotropy measurements in fiber-optic SHG microscopy to probe local molecular orientations. We have also discovered that the double-clad PCF can deliver linear excitation polarization in the core; however the SHG signal is depolarized through the inner cladding. The development of a polarization-maintained double-clad PCF would benefit the SHG polarization anisotropy measurement in compact nonlinear optical microscopy. Polarization anisotropy in fiber-optic SHG microscope and endoscopy will add a new dimension to SHG imaging for in vivo study of collagen-related diseases.
The result presented in this paper comes from the PhD work of Dr. Ling Fu at Swinburne University of Technology. Authors acknowledge support from Australian Research Council, National Natural Science Foundation of China (Grant NO. 60708025), and PCSIRT.
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