In 1995, the first author of this book joined Victoria University. Immediately after that, he established a new research group called the Optoelectronic Imaging Group (OIG), with a focus on the introduction of femtosecond lasers into optical microscopy. While the first two-photon fluorescence microscope was reported in 1990, it was not until 1996 that the first two-photon fluorescence microscope in Australia was constructed by a group of OIG Ph.D. students with a femtosecond laser supported by the major equipment fund of Victoria University. It was this new instrument that gave the OIG research students and staff a powerful tool to conduct biophotonic research. At the beginning of 2000, most of the OIG members moved to Swinburne University of Technology to form a new research centre called the Centre for Micro-Photonics (CMP). Since 1995, research students of the OIG and the CMP, including four of the authors of the book, Damian Bird, Daniel Day, Ling Fu and Dru Morrish, have made many significant contributions to femtosecond biophotonic methods. The aim of this book is to provide a systematic introduction into these methods. Chapters 1–3, 6 and 8 were completed by Min Gu and Chapters 4, 5, 7 and 9 were written by Damian Bird, Ling Fu, Dru Morrish and Daniel Day, respectively. All the authors participated in the final editing of the book.

In this chapter, we introduce a new trapping and excitation technique, which utilises a single femtosecond pulse infrared illumination source to simultaneously trap and excite a microsphere probe. The induction of morphology dependent resonance (MDR) in the trapped probe is achieved under two-photon excitation. Monitoring of the MDR in the trapped probe provides a contrast mechanism for imaging and sensing. The experimental measurement of MDR within a laser trapped microsphere excited under two-photon absorption is confirmed in Section 7.2. The effect of the laser power as well as the pulse width on the transverse trapping force is investigated in Section 7.3. The dependence of two-photon induced MDR on the scanning velocity of a trapped particle is then experimentally determined. These parameters are fundamental to the acquisition of images and sensing with femtosecond laser tweezers as described in Section 7.4.

Introduction

Laser trapping is an ideal method for the remote, non-invasive manipulation of a morphology dependent resonance microcavity. Controlled scanning and manipulation of the microcavity is possible via laser trapping. The microcavity has an enhanced evanescent field at its surface due to the resonant circumferential propagation of radiation at glancing angles greater than the critical angle. Freely suspended in a medium, the cavity becomes increasingly sensitive to its surrounding environment. The interaction of the cavity with its local environment during scanning dynamically alters the coupling to and leakage from the cavity. Monitoring the change in coupling to and leakage from the cavity over time enables imaging and sensing.

As discussed in Chapters 1 and 2, biological tissue is a highly scattering medium which will affect image resolution, contrast and signal level. This chapter discusses the effect of multiple scattering in a tissue-like turbid medium on two-photon fluorescence microscopy. Section 3.1 discusses a model based on imaging of microspheres embedded in a turbid medium. A quantitative study of the limiting factors on image quality is given in Section 3.2. In particular, the limitation on the penetration depth in turbid media, revealed from Monte-Carlo simulation and experimental measurements, is presented in Section 3.3.

Two-photon fluorescence microscopy of microspheres embedded in turbid media

Two-photon fluorescence microscopy has been extensively used due to its significant advantages over single-photon fluorescence microscopy. This technology has been used for in vivo imaging of thick biological samples. Since the required image information is taken at a large depth within a biological specimen, optical multiple scattering within tissue may result in a severe distortion on images obtained in this situation. Thus, the effect of optical multiple scattering on fluorescence image quality should be understood if high quality images are to be obtained at significant depths into a biological specimen. In this section, we present measured images of small fluorescent microspheres embedded in a turbidmedium which has different scattering characteristics under singlephoton and two-photon excitation. Imaging of small spheres embedded in a turbid medium has practical importance since it can be considered to be an approximate model of imaging small tumours embedded in biological tissue.

Ever since researchers realised that microscopy based on nonlinear optical effects can provide information that is blind to conventional linear techniques, applying nonlinear optical imaging to in vivo medical diagnosis in humans has been the ultimate goal. The development of nonlinear optical endoscopy that permits imaging under conditions in which a conventional nonlinear optical microscope cannot be used is the primary method to extend applications of nonlinear optical microscopy toward this goal. Fibreoptic approaches that allow for remote delivery and collection in a minimally invasive manner are normally used in nonlinear optical endoscopy. In Chapter 4, a compact nonlinear optical microscope based on a single-mode fibre (SMF) coupler to replace complicated bulk optics was described.

There are several key challenges involved in the pursuit of in vivo nonlinear optical endoscopy. 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 millimetre 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 flexibility and compact size to be incorporated into endoscopes to image internal organs.

The techniques introduced in Chapters 1 and 2 are emerging technologies that offer significant promise as tools for diagnostic imaging at the cellular level. Using devices founded on well established techniques such as confocal microscopy and confocal fluorescence microscopy, instruments capable of providing point-of-care pathological analysis of malignant and cancer causing tissues are becoming practical realities. Through examination of the physical properties of inherent autofluorescence or fluorescent dyes that are used as markers in conjugation with biological samples, very good detection of cellular processes can be achieved. Tagging of target biological cells makes it possible to examine cells in vivo and achieve real time three-dimensional (3D) visualisation for diagnosis of the pathological state.

However, the inherent nature of these devices is such that the conditions under which these techniques can be applied is fundamentally limited. In most cases (for definitive analysis) a surgical biopsy is performed on the patient and the sample is extensively prepared for observation by the pathologist on bulk, bench-top imaging apparatus. Ideally, examination of whole, intact specimens within internal cavities of the body would be the preferred method that may decrease patient trauma and eliminate diagnosis lag time.

One of the recent developments in confocal fluorescence microscopy is the introduction of optical fibres and fibre-optical components into the microscope geometry. Optical fibre couplers in particular offer the most compact and cost effective solution.

As discussed in Chapter 6, the trapping volume of a far-field laser trapping geometry is approximately three times larger in the axial direction than that in the transverse direction. Such trapping volume elongation leads to a significant background and poses difficulties in the observations of nano-particle dynamics. In this chapter, we deal with near-field optics using focused evanescent illumination. The recent development of near-field optical tweezers is reviewed in Section 8.1. Section 8.2 introduces the new concept of near-field laser tweezing with a focused evanescent field. This technology is characterised both experimentally and theoretically in Section 8.3. Section 8.4 presents the utilisation of a femtosecond laser beam in a near-field optical trap. Finally, some discussions on this new method are given in Section 8.5.

Near-field optical tweezers

Near-field laser trapping or tweezers means that radiation force that is used for trapping and manipulating a micro-object results from the interaction with an evanescent wave. Recently, a new trapping modality based on the evanescent wave illumination, also called near-field illumination, has been proposed and demonstrated. This trapping technique results in a significantly reduced trapping volume due to the fact that the strength of an evanescent wave decays rapidly with the distance from the surface at which the field is generated. In this section, the near-field trapping mechanism based on the different ways to generate a localised near-field is reviewed.

This chapter serves as an introduction to this book. Section 1.1 gives a brief review on the development of biophotonics and summarises the main achievements in biophotonics due to the introduction of femtosecond pulse lasers, while Section 1.2 defines the scope of the book.

Femtosecond biophotonics

Biophotonics involves the utilisation of photons, quanta of light, to image, sense and manipulate biological matter. It provides the understanding of the fundamental interaction of photons with biological media and the application of this understanding in life sciences including biological sciences and biomedicine. In that sense, biophotonics research dates back to times when biologists started to use optical microscopy and spectroscopy with a conventional light source such as a lamp. These two forms of classic biophotonic instrument revolutionised biological research and are the classic bridge between photonics and life sciences because they provide a non-destructive way to view the two-dimensional (2D) microscopic world that human eyes cannot, as well as the function of microscopic samples through colour or spectroscopic information.

Biophotonics became a recognised new discipline after the laser was invented in 1960. Laser light is fundamentally different from conventional light in the sense that it possesses high brightness in a narrow spectral window, is highly directional, and exhibits a high degree of coherence. Since 1960, these unique features have facilitated many important applications of laser technology in biological and biomedical studies. One of the important milestones in this area is the combination of laser light with an optical microscope, which led to laser scanning confocal microscopy.

The aim of this chapter is to provide a comprehensive understanding of trapped-particle near-field scanning optical microscopy (NSOM). The principle of optical trapping and laser tweezers is briefly explained in Section 6.1. Section 6.2 summarises the motivation of using a laser-trapped microsphere as a probe in NSOM. The basic principle of trapped-particle NSOM is described in Section 6.3. Two major aspects of this technique, laser trapping performance and near-field Mie scattering of dielectric and metallic particles, are discussed in Sections 6.4 and 6.5, respectively. Experimental results on image formation in trapped-particle NSOM are described in Section 6.6. In Section 6.7, some prospects for the future development of this technique are put forward.

Optical trapping and laser tweezers

Photons carry momentum. When the change in momentum occurs upon reflection, refraction, transmission and absorption of a light beam, the rate of change of momentum results in a force being exerted on an object. The origin of this force can be understood from Newton's laws. A light ray that is refracted through a dielectric particle changes its direction due to the refraction process. Since light carries momentum, a change in light direction implies that there must exist a force associated with that change. The resulting force, manifested as a recoil action due to the momentum redirection, draws mesoscopic particles toward the highest photon flux in the focal region. This recoil is unnoticeable for refraction by macroobjects such as lenses, but it has a substantial and measurable influence on mesoscopic refractive objects such as small dielectric particles.

Continued development of optical systems for simultaneous observation and manipulation of live biological specimens has produced advances in understanding cell physiology. Traditional optical microscopes have given way to multi-functional, multi-laser based observation platforms that provide us with the opportunity to interact with the specimen on a subcellular level.

This chapter gives a brief review on the development of advanced photonics technologies for biological applications including the use of femtosecond pulse lasers to interact with target cells for the stimulation of cellular responses (Section 9.1). Section 9.2 is focused on the technology of femtosecond pulse laser based microfabrication to develop microfluidic devices for applications in biology, while Section 9.3 demonstrates the use of femtosecond laser fabricated microenvironments for advanced live cell imaging of T cells. Section 9.4 discusses the use of an integrated sensor for optical sensing in microfluidic devices.

Femtosecond cell stimulation

Fluorescence signals of cells can be linked to the overall health and integrity of those cells, with fluctuations in the signals indicating effects such as changes in dye loading, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), cell activation and cell destruction. Monitoring the integrity of biological specimens that are being altered due to focused femtosecond irradiation is important to ensure no damage is being caused by such illumination.