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.

The assumptions of classical theories

The classical theory of electrolyte solutions starts with the assumption that ionic interactions in solution or between colloid particles are dictated by Coulomb, electrostatic forces alone. An ion is considered to first approximation to have a charge distribution confined to a hard sphere of a given radius. In the ‘primitive’ model the ions are immersed in water (or another solvent) within which they interact by electrostatic interactions. The solvent is treated as a passive dielectric continuum. The radius of an ion is not always just its crystallographic radius. It is an effective radius that includes one or two water layers of ‘hydration’. What occurs in the theory for the free energy of interactions involves the sum of hydrated ion radii besides the Coulomb force. The hydrated ion size is derived as a fitting parameter from comparison of theory with experiments.

For interactions between ions the long-range Coulomb interactions dominate for very dilute solutions below about 5·10−2 M, and ion size is irrelevant. At higher electrolyte concentrations, around and above about 10−1 M, the cooperative electrostatic interactions that dominate at low concentrations become progressively less important. Shorter-range forces subsumed in the ionic ‘radii’ begin to come into play. When short-range interactions between the ions become significant the molecular structure of the water in the immediate neighbourhood of the ions (hydration) becomes a dominant feature. Hydration and local hydrogen bonding are words that attempt to describe this ion-specific, local water structure induced by the ions.

Molecular forces: some of the background and history of ideas. Why molecular forces?

The matter that concerns us was most clearly articulated nearly a century ago by D'Arcy Thompson in his famous book. He reported the pleas of the early founders of the cell theory, of the then biology, and of the physiologists, that chemists should address the question of molecular forces, then unknown.

We would like to know how it is that molecular forces and the laws of statistical mechanics conspire, with the geometry of molecules and the conformations available to macromolecules, to give rise to the hierarchies of self-assembled equilibrium or dynamic steady states of matter that form cells and dictate biochemical reactivity.

In other words, the game is to link structure and function, the geometry of assemblies of molecules, to the forces that drive self assembly and recognition processes. Any insights ought to allow us to build better, useful connections between the physical and biological sciences. Despite tantalizing hints, that main aim has remained elusive.

D'Arcy Thompson tells us too that of the chemistry of his day and age, Kant said that ‘it was wisschenschaft, nicht Wissenschaft; in that the criterion of a true science lay in its reliance on mathematics’. Kant believed that Euclid's geometry was self-evidently that of nature. We now know better. Hyperbolic geometries that we shall come to later better describe the bicontinuous, random honeycombs of nature.

Cubic phases

Introduction to cubic phases

With any curved bilayer or topologically closed region such as single- or multi-walled vesicles, there is an inevitable conflict due to internal packing constraints (cf. Fig. 9.8 and 10.2).

In the energetically optimal configuration, lipids packed into the outer bilayer have normal curvature. The hydrocarbon chains are stretched. By contrast, the inner surfactants have reverse curvature. The chains are compressed. So although the interior of a bilayer can be considered fluid-like, above the Krafft (gel) temperature of the lipid it is asymmetric. As we have seen, and re-summarize, this necessary frustration has several consequences. The first is that when the radius becomes small enough, the interior layers of a multilamellar vesicle structure must collapse into aggregates different in structure to that of a bilayer. The interior can be micelles. Or it can be bicontinuous. This gives rise to supra-aggregation as a normal and expected state of self assembly. Or else the interior can be empty and surfactant-free. The second consequence is that a single-walled bilayer or vesicle has to have a radius (typically 100 nm) sufficiently large to accommodate this additional internal packing condition.

There is another way that the frustration can be relieved.

The bilayer-forming lipids or surfactants have a surfactant parameter around vH/(aPlH) ∼ 1, or <1. The average of the principal curvatures at the interface is zero for a flat bilayer.

Lifshitz theory and its extensions: an overview

Molecular recognition

The ideas behind the DLVO theory of long-range forces in colloidal particle interactions and of the electrical double layer that we have just outlined held centre stage in colloid science for at least 70 years. Quantitatively, as already remarked, agreement between experiment and theory was illusory, except at salt concentrations less than 10−2 M, or at most 10−1 M. It was illusory in the sense that while classical theory captured some essentials of the forces between ions and macromolecules, ion specificity was still missing. While the electrical double-layer forces decayed exponentially as predicted, the magnitude of the forces changed with a change in counterion or co-ion. Recall our typical examples. A change in counterion in a background electrolyte from Br− to OAc− could produce an increase in magnitude of the forces by a factor of 50 to 100! The same will be seen to occur with different surfaces with a change from Na+ to Li+. We will see much more of this specificity later. These differences depended on the nature of the charged interacting surfaces and, at the same electrolyte concentration, on the counterion. They occur even with a change in an apparently indifferent co-ion in an intervening electrolyte. The accommodation of such results could only be achieved by calling in more unquantified fitting parameters. This is unsatisfactory as predictability is lost. The attractive van der Waals forces were obscured by short-range effects due to solvent structure.

Ideas and defects of theories of self assembly

As already remarked, the first attempts to build such a unified molecular theory of self assembly that could embrace all possible aggregates began 30 years ago.

It began with Tanford's ideas on opposing intramolecular forces between surfactant molecules at interfaces.

A theory via statistical mechanics was developed based on minimal assumptions. This theory is just a characterization of self assembly. It is more complicated than, but at the same level as, nucleation theory for gas–liquid phase transitions. (Nucleation theory considers only the growth of spherical aggregates, droplets of liquid in a vapour. With surfactants we have to consider a multitude of possible aggregates of different shapes.) Nonetheless, as we have seen, the characterization that emerged gave out a set of simple design rules to predict microstructure for dilute systems.

In outline the ideas involved are these:

In the processes involved in formation of an aggregate there are several factors that can be identified.

There is a hydrophobic free energy of transfer of a monomer surfactant hydrocarbon tail, from water to the assumed bulk oil-like environment in its associated state. In an aggregate at its interface the forces between the hydrocarbon tails oppose the intra-aggregate headgroup interactions (see Fig. 10.1). These forces could be ionic, hydration, zwitterionic or steric forces. This competition at the interface sets the interfacial curvature. There is then an interfacial free energy contribution augmented by curvature contributions.

In collaboration with Drew F. Parsons.

(This chapter is more or less self-contained. It can be read independently of earlier chapters.)

Hofmeister effects in physical chemistry

Introduction

It will be clear by now that the theoretical edifice that deals with molecular forces in physical chemistry, built painstakingly over the past 150 years, is flawed. That there are defects and limitations in any theory we can live with. Of course there are. But the emerging picture from the preceding chapters is more disturbing. It seems that our intuition has become so seriously flawed that what ought to be the enabling discipline of physical chemistry has become impotent in the face of challenges posed by the biological sciences. To readers familiar with and schooled in the classical textbook literature, this claim is not easy to accept. And the burden of proof of a challenge to an established discipline lies with the proponent. So, in this chapter we recapitulate the standard literature ideas, and outline where they went off course. Then we revisit and pull together a number of examples from biology, some already mentioned, where it is clear that the standard theories fail. Then we outline our views on how it is that a reconciliation is on the way to being effected. How to fit Hofmeister effects into the scheme of things is a first issue.