Fibre-optic nonlinear optical microscopy and endoscopy

Cancer is a major health problem in the world today. Almost all cancers have a significantly better chance for therapy and recovery if detected at their early stage. The capability to perform disease diagnosis at an early stage requires high-resolution imaging that can visualise the physiological and morphological changes at a cellular level. However, resolving powers of current medical imaging systems are limited to sub-millimeter sizes. Furthermore, the majority of cancers are associated with morphological and functional alterations of cells in epithelial tissue, currently assessed by invasive and time-consuming biopsy. Optical imaging enables visualisations of tissue microstructures at the level of histology in non-invasive means. Optical imaging is suitable for detecting neoplastic changes with sub-cellular resolution in vivo without the need for biopsy. Nonlinear optical microscopy based on multi-photon absorption and higher harmonic generation has provided spectacular sights into visualisation of cellular events within live tissue due to advantages of an inherent sectioning ability, the relatively deep optical penetration, and the direct visualisation of intrinsic indicators. Two-photon excited uorescence (TPEF) from intrinsic cell components and second harmonic from asymmetric supermolecular structures can provide complementary information regarding functionalities and morphologies in tissue environments, thus enabling premalignant diagnosis by detecting the very earliest changes in cellular structures. During the past sixteen years, nonlinear optical microscopy has evolved from a photonic novelty to a well-established laboratory tool. At present, in vivo imaging and long-term bedside studies by use of nonlinear optical microscopy have been limited due to the fact that the lack of the compact nonlinear optical instrument/imaging technique forces the performance of nonlinear optical microscopy with bulk optics on the bench top. Rapid developments of fibre-optics components in terms of growing functionalities and decreasing sizes provide enormous opportunities for innovation in nonlinear optical microscopy. Fibre-based nonlinear optical endoscopy will be the soul instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible with a conventional optical microscope. Lots of efforts have been made for development of miniaturised nonlinear optical microscopy. However, there are major challenges remaining to create a nonlinear optical endoscope applicable within internal cavities of a body. First, an excitation laser beam with an ultrashort pulse width should be delivered efficiently to a remote place where efficient collection of faint nonlinear optical signals from biological samples is required. Second, laser-scanning mechanisms adopted in such a miniaturised instrumentation should permit size reduction to a millimeter scale and enable fast scanning rates for monitoring biological processes. Finally, the design of a nonlinear optical endoscope based on micro-optics must maintain great exibility and compact size to be incorporated into endoscopes to image internal organs. Although there are obvious difficulties, development of fibre-optic nonlinear optical microscopy/endoscopy would be indispensible to innovate conventional nonlinear optical microscopy, and therefore make a significant impact on medical diagnosis. The work conducted in this thesis demonstrates the new capability of nonlinear optical endoscopy based on a single-mode fibre (SMF) coupler or a double-clad photonic crystal fibre (PCF), a microelectromechanical system (MEMS) mirror, and a gradientindex (GRIN) lens. The feasibility of all-fibre nonlinear optical endoscopy is also demonstrated by the further integration of a double-clad PCF coupler. The thesis concentrates on the following key areas in order to exploit and understand the new imaging modality. It has been known from the previous studies that an SMF coupler is suitable for twoii photon excitation by transmitting near infrared illumination and collecting uorescence at visible wavelength as well. Although second harmonic generation (SHG) wavelength is farther away from the designed wavelength of the fibre coupler than that of normal TPEF, it is demonstrated in this thesis that both SHG and TPEF signals can be collected simultaneously and e ciently through an SMF coupler with axial resolution of 1.8 um and 2.1 um, respectively. The fibre coupler shows a unique feature of linear polarisation preservation along the birefringent axis over the near infrared and the visible wavelength regions. Therefore, SHG polarisation anisotropy can be potentially extracted for probing the orientation of structural proteins in tissue. Furthermore, this thesis shows the characterisation of nonlinear optical microscopy based on the separation distance of an SMF coupler and a GRIN lens. Consequently, the collection of nonlinear signals has been optimised after the investigation of the intrinsic trade-off between signal level and axial resolution. These phenomena have been theoretically explored in this thesis through formalisation and numerical analysis of the three-dimensional (3D) coherent transfer function for a SHG microscope based on an SMF coupler. It has been discovered that a fibreoptic SHG microscope exhibits the same spatial frequency passband as that of a fibreoptic reection-mode non-uorescence microscope. When the numerical aperture of the fibre is much larger than the convergent angle of the illumination on the fibre aperture, the performance of fibre-optic SHG microscopy behaves as confocal SHG microscopy. Furthermore, it has been shown in both analysis and experiments that axial resolution in fibre-optic SHG microscopy is dependent on the normalised fibre spot size parameters. For a given illumination wavelength, axial resolution has an improvement of approximately 7% compared with TPEF microscopy using an SMF coupler. Although an SMF enables the delivery of a high quality laser beam and an enhanced sectioning capability, the low numerical aperture and the finite core size of an SMF give rise to a restricted sensitivity of a nonlinear optical microscope system. The key innovation demonstrated in this thesis is a significant signal enhancement of a nonlinear optical endoscope by use of a double-clad PCF. This thesis has characterised properties of our custom-designed double-clad PCF in order to construct a 3D nonlinear optical microscope. It has been shown that both the TPEF and SHG signal levels in a PCF-based system that has an optical sectioning property for 3D imaging can be significantly improved by two orders of magnitude in comparison with those in an SMF-based microscope. Furthermore, in contrast with the system using an SMF, simultaneous optimisations of axial resolution and signal level can be obtained by use of double-clad PCFs. More importantly, using a MEMS mirror as the scanning unit and a GRIN lens to produce a fast scanning focal spot, the concept of nonlinear optical endoscopy based on a double-clad PCF, a MEMS mirror and a GRIN lens has been experimentally demonstrated. The ability of the nonlinear optical endoscope to perform high-resolution 3D imaging in deep tissue has also been shown. A novel three-port double-clad PCF coupler has been developed in this thesis to achieve self-alignment and further replace bulk optics for an all-fibre endoscopic system. The double-clad PCF coupler exhibits the property of splitting the laser power as well as the separation of a near infrared single-mode beam from a visible multimode beam, showing advantages for compact nonlinear optical microscopy that cannot be achieved from an SMF coupler. A compact nonlinear optical microscope based on the doubleclad PCF coupler has been constructed in conjunction with a GRIN lens, demonstrating high-resolution 3D TPEF and SHG images with the axial resolution of approximately 10 m. Such a PCF coupler can be useful not only for a fibre-optic nonlinear optical probe but also for double-clad fibre lasers and amplifiers. The work presented in this thesis has led to the possibility of a new imaging device to complement current non-invasive imaging techniques and optical biopsy for cancer detection if an ultrashort-pulsed fibre laser is integrated and the commercialisation of the system is achieved. This technology will enable in vivo visualisations of functional and morphological changes of tissue at the microscopic level rather than direct observations with a traditional instrument at the macroscopic level. One can anticipate the progress in bre-optic nonlinear optical imaging that will propel imaging applications that require both miniaturisation and great functionality.