1. I will arise and go now, for always night and day

2. I hear lake water lapping with low sounds by the shore;

3. While I stand on the roadway, or on the pavements gray,

4. I hear it in the deep heart’s core.

5. (William Butler Yeats, The Lake of Innisfree, 1888)

GENERAL IDEA

Here we venture beneath the surface of the cell membrane to explore some of the key biological processes that occur in the core of cells, which have been investigated using single-molecule biophysics techniques either in living samples or in physiologically relevant settings in the test tube.

Introduction

As we saw in the previous chapter, the cell membrane, with its various associated integrated protein complexes, is a key structure being the first point of contact for the cell with the outside world. However, the meat of the cellular machinery for metabolizing nutrients, manufacturing new molecular material, responding to signals detected at the cell membrane surface and for storing its genetic code are all located in the innards of the cell, either occurring in the cytoplasm often associated with a variety of cellular sub-structures or, in the case of eukaryotic cells, in specialized membrane-bound organelles. Previously, we discussed some details of two such organelles in the context of membrane-localized processes, the chloroplasts that perform photosynthesis in plant cells and mitochondria that generate the universal cellular fuel of ATP. Here, we will also extend the discussion to processes occurring in the cell nucleus, how the genetic code is packaged and ultimately replicated, and the means by which this code is converted into molecules of protein. But we will begin the chapter outside the nucleus and discuss the biophysical properties of the cytoplasm, and the mechanisms by which molecular cargo is controllably trafficked and sorted inside the cell.

GENERAL IDEA

Here we discuss the miscellaneous experimental techniques that allow us to monitor single biological molecules using physical approaches which do not rely primarily on visible light.

Introduction

There now exist several methods which permit measurement of the presence of single biological molecules using physical principles which do not rely primarily on the detection of visible light. These include a variety of scanning probe microscopy techniques, including atomic force microscopy, which are discussed in detail in the first section of this chapter. In addition, significant advances in our understanding of single-molecule biology have come from methods using electron microscopy, which is one of the pioneering techniques used for obtaining structural information on fixed single-molecule samples. Recent advances in the measurement of small ion currents through both solid-state and native physiological nanometre length scale pores have furthered our knowledge of many areas of single-molecule bioscience. Furthermore, Raman spectroscopy has now advanced to a level of sensitivity such that measurements of single biological molecules are feasible. And finally, there are several microscopy methods which allow us to deduce the position of single molecules using primarily infrared optical tweezers.

GENERAL IDEA

Here we take stock of the remarkable developments and innovations in biophysics that have allowed us to address very challenging and fundamental questions about the key cellular processes, and speculate where this might lead in the near future.

Introduction

The emergence of single-molecule cellular biophysics represents a coming-of-age of single-molecule bioscience. The first generation of single-molecule experiments resulted in some exceptionally pioneering developments in terms of the physics of techniques and novel analytical methods and in terms of significantly increasing our understanding of the functioning of isolated biological molecules. But now, as we have seen from myriad investigations discussed in this book, the next generation of single-molecule bioscience has opened up outstanding opportunities to study biological processes under physiologically highly appropriate conditions – in other words to gain enormous insight into how single molecules really function in the context of their native environment of the living cell. In this final chapter we survey the developments that have led us to this point, and ask the question ‘what next?’ As we will see, there is great potential to apply these novel technologies in areas that may have a large future impact on society, namely those of bionanotechnology and synthetic biology, fuel production for commerical use and single-molecule biomedicine.

GENERAL IDEA

Here we outline some of the key concepts and terminology in cell and molecular biology to orientate readers from a more physical science background.

Introduction

The classical biological view is that living organims are typically structured in a hierarchical manner in terms of physiological function relating to length scale. For example, a complex multi-cellular organism is composed of smaller units called organs which appear to be dedicated primarily to a subset of biological processes, and these may be further deconstructed into different tissues, and these tissues may be further sub-divided into smaller structural features consisting ultimately of individual cells, or some structural matrix secreted by cells. Single cells, whether part of a multi-cellular organism as in the human body or simply the organism itself as for unicellular life forms such as bacteria, can in turn be broken up conceptually into smaller subunits. In essence these are structural sub-cellular features which appear to work together to perform a narrow subset of biological functions, for example cell organelles such as the nucleus in certain cell types. Ultimately, smaller sub-cellular feautures can be perceived as collections of single biological molecules.

GENERAL IDEA

In this chapter we encounter the biophysical methods which can be used to measure forces exerted by single biological molecules, and also techniques which can allow us to manipulate single molecules controllably.

Introduction

There now exist several methods which permit highly controlled measurement and manipulation of the forces experienced by single biological molecules. These varied tools all come under the banner of force transduction devices, since they convert mechanical molecular forces into some form of amplified, measurable signal. They share other common features, for example, in general, the single molecules are not manipulated directly but are in effect physically conjugated, usually via one or more chemical links, to some form of adapter which is the the real force transduction element in the system. The principal forces which are used to manipulate the relevant adapter include optical, magnetic, electrical and mechanical forces, and in general all these forces are implemented in an environment of complex feedback electronics and stable, noise-minimizing microscope stages, for the purposes of both measurement and manipulation.

GENERAL IDEA

In this chapter we discuss the techniques which are available to the experimental scientist who wishes to visualize or detect single biological molecules primarily using visible light, both in the test tube and in the living cell.

Introduction

How can we ‘see’ something like a single biological molecule, which is of the order of a thousand million times smaller than a typical object in the macroscopic world that we visualize with our naked eyes? The region of the human eye responsible for detecting light, the retina, consists of two types of cells called rods and cones (rods differ by being 100 times more sensitive than cones but they respond more slowly, have less spatial resolution and do not discriminate colour), both of which can convert detected photons of light into electrical signals, conveyed via ion channels and nerve fibres into the brain. The resolving power of the human eye, the visual acuity, is a measure of the smallest angular separation that the eye can resolve, which for humans has a theoretical limit equivalent to ~0.01°, about 20 milliradians, determined by the limit of optical diffraction set by the wavelength of the incident light and the diameter of the aperture in front of the ‘imaging device’ (here set by the pupil of the eye), as shown in Figure 3.1A.