Bacteria—plant associations

The origin and evolutionary development of higher plants has occurred in environments that were already colonised by bacteria, resulting in the co-evolution of a range of bacteria—plant associations. The associated microbes may be broadly considered in two main categories: epiphytic bacteria (present on the outside of the plant) and internal bacteria (infecting the plant tissue).

Epiphytic bacteria

These are associated with the plant surface, which is generally divided into root (rhizosphere) and aerial (phyllosphere) regions. A wide range of bacteria are adapted to various microenvironments at the soil and air interface, and are important in such aspects as nutrient uptake, frost damage, and biological control of plant pathogens. Many of these epiphytic bacteria are saprophytes, obtaining complex nutrients from the plant. Some epiphytic bacteria are also parasites, spending part of their life cycle on the plant surface, and part within the plant tissue.

Infective bacteria: parasites and symbionts

Parasitic bacteria are able to invade plant tissue, where they grow and multiply, and cause localised or general deterioration in the health of the plant. The great majority of these parasites are extracellular, multiplying within intercellular spaces but not penetrating plant cell walls or entering protoplasts. The relatively few parasitic bacteria that are able to penetrate the higher plant cell include members of the genus Agrobacterium (with the ability to transfer part of the genome into the plant cell) and Rhizobium (where the whole organism enters the plant cell).

Summary

X-ray microanalysis has developed into a reliable technique for the determination of the intracellular distribution of elements, due to the use of cryopreparation techniques that preserve the in vivo localisation of diffusible elements, and the introduction of quantitative techniques. X-ray microanalysis is now used for investigating a great number of biological problems. The major fields of application are environmental biology/toxicology and physiology/pathology. In human and animal physiology (and pathology), studies have concentrated on the role of calcium in cellular processes; but ion transport in epithelia, under both normal and pathological conditions, is also an important field of study. Furthermore, the possible relation of Na+ ions to mitogenesis and oncogenesis is a problem where X-ray microanalysis has made interesting contributions.

Introduction

Biologists have been using electron probe X-ray microanalysis for the past 30 years to complement the morphological information provided by the electron beam with the chemical information provided by the process of X-ray generation in the specimen. The first applications were carried out using a crystal spectrometer, but when energy-dispersive (semiconductor-based) spectrometers became available, these soon became the dominating type of instrument for biological X-ray microanalysis. Energy-dispersive spectrometers were easier to handle, allowed the analysis of all elements simultaneously, and had a better efficiency, which allowed lower beam currents. Only in the analysis of microdroplets (Roinel, 1988) is the wavelength-dispersive spectrometer the instrument of choice.

X-ray microanalysis has become increasingly used to study the elemental composition of biological specimens and has provided valuable information on the identity and occurrence of diffusible and bound ions, inorganic deposits and elements that are structural components of macromolecules. The attractions of X-ray microanalysis to the potential investigator in terms of spatial resolution and range of elements that can be detected have already been mentioned in Section A. The analytical sensitivity in terms of minimal detectable mass (10−19 g) also presents considerable opportunity to the analyst, though the value for sensitivity on a mass fraction basis (normally no better than 0.1 % with energy dispersive systems) poses major limitations.

The diversity of applications can be considered both in terms of general areas of study and type of specimen examined.

Applications of X-ray microanalysis to different subject areas of biology

X-ray microanalysis has been used in a variety of disciplines within biology, as exemplified by the range of chapters in this section of the book. These areas include microbiology, plant biology, animal biology (vertebrate and invertebrate tissues, cultured animal cells), medicine and environmental biology. A computer survey of the relative numbers of papers in these different areas (Table D1) indicates a clear preponderance in the spheres of animal biology and medicine.

The role of X-ray microanalysis in human physiology and medical research has recently been reviewed by Shelburne et al. (1989), and is important in the study of aspects such as the activity of Ca in cellular processes, ion transport in epithelia and the possible importance of cations in mitogenesis and oncogenesis.

Summary

Electron energy-loss spectroscopical (EELS) data and those acquired by electron probe X-ray microanalysis (XRMA) add chemical information to morphological structures in electron microscopical images. Recent instrumental developments lead to the need for a comparison of their various microanalytical potentials in relation to biological materials, especially their possibilities for quantitative image analysis. The aim of this chapter is to describe the present state of the art in electron energy-loss image analysis and to compare the results with those from previous studies on images of the same material with electron probe X-ray microanalysis. The acquisition of digitised electron spectroscopic images (ESI) from ultrathin sectioned cells and tissues allows the simultaneous morphometric and chemical analysis of such material. A recurring problem in the analysis is the segmentation of the images. It is shown that segmentation can be achieved by thresholding, using the first derivative of the grey-value frequency histograms constructed from such images. This method applies to electron energy-loss images as well as to images acquired by X-ray microanalysis. The influence of the image contrast and of the image averaging procedure during acquisition on the morphometrical parameters and perimeter is indicated. It is demonstrated that quantification of element-related spectra and net-intensity images is possible. One of the advantages of digitised images, to show the co-localisation of several elements within a single organelle, is illustrated. An example of element concentration determination using co-embedded Bio-standards present in the same section as the ‘unknown’ is given.

X-ray mapping is now an important qualitative and quantitative aspect of X-ray microanalysis of biological specimens, and has been the subject of a number of recent research papers and review articles (Chang, Shuman & Somlyo, 1986; Fiori, 1986; Fiori et al., 1988; Ingram et al., 1987; Lamvik et al., 1989; Sauberman & Heyman, 1987). The related technique of electron energy loss mapping has also been the subject of much recent interest (Jeanguillaume et al., 1983; Leapman & Ornberg, 1988) and is discussed separately by de Bruijn et al. in this volume.

X-ray mapping is the technique whereby an image is formed using the X-ray signal from a specified energy range, in order to show elemental distribution within the sample. X-ray mapping in its simplest form has been available since the early days of X-ray microanalysis. The technique has evolved in sophistication over the past 20 years, the main milestones being the development of digital mapping in the late 1970s and quantitative mapping in the late 1980s. The methods currently available are:

1. (a) analogue dot mapping,

2. (b) qualitative digital mapping,

3. (c) quantitative digital mapping.

Analogue dot mapping

This procedure was developed in the mid 1960s. Dot maps are formed when X-rays generated within a specified energy range appear as bright dots on the electron microscope display as the electron beam scans the sample. The advantage of the technique is that maps are very quick to set up and acquire.

Introduction

Determination with an electron probe of the elemental composition of liquid samples of ‘small’ volumes is not microanalysis sensu stricto. Indeed, these volumes are in the range of 10−10 to 10−11 litre, i.e. still several orders of magnitude larger than the 1 μm3 volume normally excited in conventional X-ray microanalysis. However, dealing with nanolitre volumes is a common situation for a biologist, who then faces the problem of determining a number of elements in so ‘small’ a volume. This explains why the technique of X-ray analysis of droplets has been so extensively used over the past two decades. This chapter will not focus on the technical details of sample preparation. Two recent review papers (Quamme, 1988; Roinel, 1988) have abundantly dealt with this problem. After a brief overview of the technique (sample preparation and characteristics, sensitivity, accuracy and precision), we will show how efficient the use of the technique has been in renal physiology, especially in vivo for the study of the handling of magnesium by the kidney and in vitro for studies involving the microperfusion of isolated tubules.

Although the droplet technique has so far mainly been used in investigations of the renal physiology of mammals, it has also been applied to the study of the excretory function of insects, and to the study of other physiological functions in man (Ferrary et al., 1988), mammals and various animals (see bibliographic reviews in Roinel & Rouffignac, 1982, and more recently Quamme, 1988).

Introduction

General applications of the technique to bacterial cells

Electron probe X-ray microanalytical studies have been carried out on a wide range of bacterial cells, using a variety of preparation techniques. These studies have involved many different applications and have relevance to areas of environmental microbiology, studies on cultured cells, human pathology, plant pathology, metal toxicity and biochemical research. Some of these studies are summarised in Table 12.1, which emphasises the broad contribution that this technique has made to bacteriology.

The main purpose of this chapter is to explore the potential uses of electron probe X-ray microanalysis in bacterial research, and to consider the different experimental approaches that can be used with these cells. The chapter will concentrate particularly on bacterial pathogens of higher plants, since this has been an area of special interest in this laboratory.

Elemental composition of plant pathogenic bacteria

Plant pathogenic bacteria are facultative parasites, able to grow and multiply both outside and inside the plant – where they may cause disease. The elemental composition of these organisms is of considerable interest in relation to a number of key aspects of their existence (Sigee, Hodson & El-Masry, 1989). Changes in the concentration of specific cations, in particular, have been implicated in various functional processes – including hydrolysis of bacterial toxins (Levi & Durbin, 1986), interactions between microorganisms at the plant surface (De Weger et al., 1988) bacterial changes during disease (El-Masry & Sigee, 1989), chemical control measures (Sekizawa & Wakabayashi, 1990) and in vitro effects of heavy metals (Sigee & Al-Rabaee, 1986; Hodson & Sigee, 1991).

The technique of electron probe X-ray microanalysis (XRMA), by which the elemental composition of specimens can be determined on a microscopical scale, has now been applied to biological materials for about 30 years, and in that time has made many valuable contributions to a variety of biological problems. To improve awareness of the technique, and to provide a discussion forum for those interested in biological XRMA, the Biological X-ray Microanalysis Group was formed in Britain some five years ago. Although its primary purpose was to hold regular meetings, the possibility of producing a book which reviewed both developments in the equipment and the wide variety of applications for which XRMA could be used was another aim, now realised in the present volume. This book arose out of a meeting held by the Biological X-ray Microanalysis Group in Manchester, England, in April 1991. What we have tried to produce is not simply another set of conference proceedings, but a well balanced and integrated series of chapters describing the hardware and software used for XRMA, the necessary procedures for specimen preparation and quantification, and the enormous range of applications of the technique in biology.

It might be supposed that after 30 years' application of biological X-ray microanalysis, the field might be settling down to a comfortable but perhaps rather routine maturity. The contributions in this book show, however, that this is far from being so, with many exciting developments in all aspects of the subject.

Introduction

Cell cultures are frequently used in biological studies because it is possible to control closely the experimental conditions. Several different in vitro systems are currently used in biochemical and morphological studies of the effects of growth factors, teratogens, toxic substances and heavy metals on cell physiology and morphology. Changes in the elemental composition of the cells can be measured by X-ray microanalysis (XRMA) which, combined with biochemical or morphological techniques, contributes to an understanding of the role of ions in physiological and pathological processes. XRMA has several important advantages over more widely used analytical methods, e.g. atomic absorption or flame spectrophotometry (Wroblewski et al., 1989). It allows analysis of individual cells in a heterogeneous cell population, which cannot be achieved by the above mentioned methods. Cell cultures can be relatively easily prepared for XRMA without the use of costly equipment such as a cryostat or cryoultramicrotome. There is no need for dissection (compared to tissue samples) and the thickness of cultured monolayers, which is in the range of 5 μm, rarely exceeding 10 μm, improves conditions for good fixation by quench freezing. Due to the ease of specimen preparation, several XRMA investigations have been carried out on cultured cells (Lechene, 1989; Saubermann & Stockton, 1988; Wroblewski et al., 1983b; Wroblewski & Roomans, 1984, Wroblewski et al., 1987; Zierold, Gerke & Schmitz, 1989). Intracellular element localisation, epithelial transport, dynamic processes and pathological accumulation of extraneous and endogenous material have been studied.

X-ray microanalysis of plant cells

Ions play vital roles in the water relations of plant and animal cells and in the regulation of cellular metabolism. An important component of the function of these elements is often a change in subcellular distribution. Investigation of the subcellular distribution of elements such as sodium, potassium, calcium, magnesium and chlorine, which are highly mobile in the aqueous phase, is a most challenging problem in biology. In order to study the distribution and transport of elements within cells a technique with high sensitivity and high resolution is required. The chosen method must not, in itself, alter the native elemental distribution during sample preparation or during measurement. Measurement of the (inorganic) elemental content of compartments (cell walls, cytoplasm and vacuole) within plant cells can be approached by a number of different methods, including the analysis of efflux of radioactive ions, analysis of tissues with different compartmental volume fractions, the use of microelectrodes specific to particular ions, by NMR in some instances and by X-ray microanalysis of individual cells. Of these, X-ray microanalysis offers more than any of the other methods in terms of both resolution and sensitivity. The use of microelectrodes is restricted in plant biology because of the physical strength of the walls of many cell types, and the difficulties of visualising the electrode tip in intact tissues in other than surface cells. As a technique, X-ray microanalysis also has the potential to resolve concentration differences within the cytoplasmic phase (e.g. between different organelles).

The suitability of X-ray microanalysis for histochemistry

The great majority of applications of X-ray microanalysis (XRMA) in biology may be regarded as histochemical, in the sense that they are studies of the chemical composition of cells or tissues in situ. Conventionally, however, the term histochemistry is used to denote the investigation of the chemistry of cells and tissues in situ using specific reactions that produce a distinctive reaction product that can be detected by some microscopical method. For light microscopy, a coloured reaction product is normally used. Although this approach has been extremely successful, not every cellular substance of interest can be identified in this way. In addition, coloured reaction products may diffuse, or dissolve in mountants, and can rarely be quantified satisfactorily. For electron microscopy, reliance is placed on reaction products which are electron dense. As well as the problems of diffusion and quantification noted above for light microscopical histochemistry, it may also be difficult at times to distinguish the reaction product from the normal staining of the section. Alternatively, if the section is left unstained, the histochemical reaction product may be clearly visible, but difficult to localise because of the low contrast of the rest of the material. X-ray microanalysis has the potential to help with all these problems.

Problems of diffusion of histochemical reaction products are likely to be greatest in multi-step reactions, such as those for many enzymes.

There is considerable interest in the application of X-ray microanalysis to the study of biological specimens since this is one of the few techniques which allows the study of diffusible elements at a subcellular level. Although it is easy to obtain element ratios from spectra produced by X-ray microanalysis, fully quantitative data are required for the results to take their place alongside those obtained by use of other physiological techniques. Quantitative data are still published routinely from only a small number of laboratories, implying that some difficulties may be encountered in the application of quantification to biological specimens. Here, the two main methods used for obtaining quantitative results from the analysis of thin sections will be outlined briefly; details of other methods can be found in Hall (1989a). The discussion will concentrate on the appraisal and interpretation of results as this is an area where problems may arise.

The preparative steps involved in the study of diffusible elements are, firstly, cryofixation (reviewed by Zierold, this volume), which is usually followed by cryosectioning and analysis of the sections in the frozen–dried state (the sections can be analysed frozen–hydrated, but this is technically much more difficult – see Warley & Gupta, 1991). As an alternative the cryofixed specimen may be freeze dried and embedded, or embedded using low temperature techniques. The initial part of this discussion will focus on the quantification of data obtained from the analysis of frozen–dried cryosections; in a later section the problems encountered when applying quantitation to embedded material will be highlighted.

Introduction

The use of a focused beam of high energy (MeV) protons to image and analyse thin samples offers many unique advantages over existing techniques. As shown in Fig. 5.1, the interaction of the proton beam with matter can take many forms. Non-nuclear reaction products such as characteristic X-rays, secondary electrons, backscattered protons and nuclear reaction products such as gamma rays and nuclear particles, can provide a wealth of analytical information on the sample under investigation. If the sample is thin enough to allow the protons to pass through, then the subsequent detection of the transmitted protons allows structural information to be obtained.

Described here is an example of the use of three of the techniques associated with the proton beam interaction, as applied to Alzheimer's Disease tissue. These techniques, utilising the detection of characteristic X-rays, back-scattered protons and transmitted protons, can be simultaneously applied to form a powerful array of nuclear particle based techniques which we have called nuclear microscopy.

Nuclear microscopy: techniques for analysis and features of the scanning proton microprobe

Proton induced X-ray emission (PIXE)

When a particle collides with an atom, the probability of knocking out an inner core electron is optimised when the velocity of the incoming particle matches the velocity of the inner core electron. This occurs at keV energies when electrons are used as the impinging particle, and at MeV energies when protons are used.

Introduction

Invertebrates constitute 90% or more of all the living species in the Animal Kingdom. The class Insecta alone has more than one million recorded species. Invertebrate species can be found flourishing in virtually every ecological environment from the deepest of oceans in the vicinity of hydrothermal vents to the highest of mountains. This process of speciation during the course of evolution has not only generated a bewildering range of morphological form, size and shape but has also led to an exploitation of every conceivable physico-chemical principle in the physiological adaptation. The celebrated Danish zoophysiologist August Krogh once said that there is always some organism which has maximally expressed a particular biological phenomenon and therefore would be ideally suited for investigating that principle. Similarly, while promoting the use of insects in the study of general physiology, Wigglesworth (1948) has said: ‘Insects … are so varied in form, so rich in species, and adapted to such diverse conditions of life that they afford unrivalled opportunities for physiological study’. Furthermore, numerous invertebrate species can often be obtained in large number and maintained in the laboratory at a relatively low cost.

A perusal of any modern text-book of cell and developmental biology, comparative physiology etc. would show that over the past century most of the fundamental biological concepts were founded by investigating material from some invertebrate species. The diffusible ions Na, K, Cl, Mg, Ca, Zn and also H, OH are now implicated in virtually all the cell functions.

Introduction

Quantitation routines which have been developed for use in materials science cannot usually be applied to biological specimens because biological material has special characteristics.

1. (a) It usually consists of elements of interest such as sodium, magnesium, potassium etc., which are present at low concentrations (less than 5%) in an organic matrix of the low atomic number elements carbon, hydrogen, oxygen and nitrogen. These matrix elements are not detected by the usual beryllium window detector. Because of this composition, standards are required when biological material is analysed.

2. (b) The major component of living tissue is water. This is generally removed during specimen preparation, though hydrated tissue can be studied if a suitable cold stage is available.

3. (c) Biological specimens often present a rough surface and are of uneven thickness.

Because of these factors, software for the application of quantitation to biological specimens must include routines which are able to cope with the differences in specimen thickness that are encountered (e.g. using the Hall method) and also offer routines for the estimation of local water content. In addition, the programme should be easy to use and should be capable of processing spectra automatically when large amounts of data are being collected (as is the case with biological specimens).

Introduction

Biomaterials include all materials that are used as surgical implants, dental materials and prosthetic implants. The applicability of a material as a biomaterial is determined by its chemical and mechanical properties, by its behaviour in contact with body fluids, cells and tissues, and by the eventual release of toxic components during biodegradation.

Biomaterials science is, therefore, concerned with tissue reactions that occur when artificial, manufactured devices are brought into contact with the living body.

Various microscopical techniques, including light microscopy, transmission electron microscopy and scanning electron microscopy are applied to study these tissue reactions. X-ray microanalysis is of special value in studies on phenomena occurring during biodegradation, i.e. the release and accumulation of chemical trace elements, and also to study the process of calcium phosphate deposition in bone bonding implants. In this chapter, the role of X-ray microanalysis in biomaterials research is discussed, specifically in reference to the biological evaluation of materials in a tissue culture system and post-implantation in experimental animals.

The total alloplastic middle ear

Implants routinely consist of various types of biomaterial, as shown, for example, by the artificial Total Alloplastic Middle Ear implant (TAM) (Bakker, 1988).

The TAM was developed to reconstruct in one operation the canal wall, the tympanic membrane and the ossicle chain in patients that suffer from chronic otitis media. It consists of a hydroxyapatite canal wall as suspension system to which a hydroxyapatite ossicular chain is connected.

Summary

Radiation damage in the electron microscope is reviewed in the context of biological X-ray microanalysis. A phenomenological description of the dependency of radiation induced mass loss on electron dose in frozen–hydrated and frozen–dried biological material is given and some semiquantitative data are presented as a rule of thumb for practical X-ray microanalysis. Quantitative imaging, spatial deconvolution and extrapolation to zero dose are discussed as possibilities to minimise radiation effects in low temperature X-ray microanalysis. The consequences for the measurement of local water fractions by low temperature electron microscopy and X-ray microanalysis are deduced.

Introduction

X-ray microanalysis can be a very powerful tool in physiological research, because of its ability to track with high resolution the distribution of ions in cells and tissues. This requires low temperature techniques for three reasons. First, there is a beneficial effect of low temperatures in terms of radiation damage. Second, the retention of water during the measurement excludes a number of possible redistribution artefacts. Third, retention of water by cooling is required by most of the techniques to measure local water fractions.

It was clear from the beginning of biological X-ray microanalysis that its potential could be fully exploited only when the distribution of water as well as the distribution of detectable elements could be measured (see, for instance, Gupta, Hall & Maddrell, 1976).