Cover

Half-title

Title

Copyright

Contents

List of contributors

Preface

SECTION A DETECTION AND QUANTIFICATION OF X-RAYS

References

1 Recent developments in X-ray detectors and their relevance in biological X-ray microanalysis

A Introduction

B X-ray generation

C X-ray detection

1 Si{Li) versus HpGe crystal

2 Crystal size

3 Window choice

(i) BERYLLIUM WINDOW

(ii) ULTRATHIN WINDOW AND WINDOWLESS

(Hi) ONE ATMOSPHERE WINDOW

D X-ray analysis in biological research

E Conclusion

References

2 Quantitative software for biological applications of X-ray microanalysis

A Introduction

B Application of the Hall method (continuum narmalisation)

C Corrections for continuum intensity

D Measurement of local water concentrations

1 Comparison offrozen-hydrated and frozen-dried specimens

2 Measurement of oxygen concentration

E Worked examples

1 Operation of programme - calibration and S-factors

2 Results

Acknowledgement

References

3 X-ray mapping techniques in biology

A Analogue dot mapping

1 Dot mapping low concentrations

2 Dot mapping rough surfaces

B Qualitative digital mapping

1 Processing low concentration X-ray maps

2 Processing maps from rough specimens

C Quantitative mapping

1 Coping with drift

(i) SPECIMEN DRIFT

(ii) SPECTROMETER DRIFT

2 Information generated from quantitative maps

(i) STANDARD DEVIATION MAPS

(ii) FIT INDEX MAPS

(iii) QUANTITATIVE MAPS FROM BIOLOGICAL THIN SECTIONS

(iv) QUANTIFICATION OF SELECTED REGIONS

D Conclusions

References

4 Quantitative X-ray microanalysis of thin sections in biology: appraisal and interpretation of results

A Obtaining quantitative results from the analysis of frozen-hydrated or frozen-dried specimens

1 Measurement of elemental mass per unit mass {continuum normalisation)

2 Measurement of mass per unit volume {characteristic alone)

B Standards

C Appraisal and interpretation of results

D Measurement of water content

E Quantification of results from resin-embedded specimens

F Conclusions

Acknowledgements

References

SECTION B ASSOCIATED TECHNIQUES

References

5 Proton probe microanalysis in biology: general principles and applications for the study of Alzheimer's Disease

A Introduction

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

1 Proton induced X-ray emission (PIXE)

2 Rutherford backscattering spectrometry (RBS)

3 Scanning transmission ion microscopy (STIM)

4 Specimen preparation

5 The nuclear microscope

C The use of nuclear microscopy in Alzheimer's Disease

1 General features of the disease

2 Identification of senile plaques using STIM

3 Analysis of senile plaques using PIXE and RBS

D General conclusions

Acknowledgements

References

6 Electron energy-loss spectroscopy and electron probe X-ray microanalysis of biological material: a comparative quantitative analysis of electron microscopical images

A Summary

B Introduction

1 General comparison between electron energy-loss and X-ray microanalysis of biological materials

2 Elemental mapping and image analysis by electron energyenergyloss and electron probe X-ray microanalysis

C Materials and methods

D Results

1 Morphometric analysis ofultrathin sectioned cells by electron spectroscopic imaging

(i) THE ACQUISITION OF AREA AND/OR PERIMETER

(ii) THE ACQUISITION OF THE AREA FRACTION PER CELL CROSSSECTION

(iii) ACQUISITION OF THE AREA FRACTION PER STANDARD TISSUE AREA

2 Quantitative spectral and image analysis

(i) QUANTITATIVE SPECTRAL ANALYSIS

(ii) QUANTITATIVE IMAGE ANALYSIS

(iii) IMAGE INTEGRATION AND CO-LOCALISATION

3 The use of ultrathin-sectioned Bio-standard material

(i) IN SPECTRAL ANALYSIS

(ii) IN IMAGE ANALYSIS

E Discussion

1 Morphometry

2 Quantitative spectral analysis

3 Background correction in element-related images

4 Co-embedding of Bio-standards

5 Comparison of energy-dispersive X-ray and electron energyloss spectroscopic spectra

6 Pre- versus post-acquisition digitation

(i) RESOLUTION

(ii) TOTAL ACQUISITION TIME

(iii) X-RAY YIELD VERSUS ELECTRON YIELD

References

SECTION C SPECIMEN PREPARATION

Selected reading material

7 Rapid freezing techniques for biological electron probe microanalysis

A Introduction

B Preparation paths for electron probe microanalysis

C Rapid freezing techniques

D Rapid freezing of functional states

E Rapid freezing of dynamic processes

F Conclusion

Acknowledgement

References

8 Radiation damage and low temperature X-ray microanalysis

A Summary

B Introduction

C Radiation damage: specimen preparation and general operational parameters

1 Relocation artefacts

2 Mass loss

D Further techniques to minimise radiation damage effects during low temperature X-ray microanalysis

E Consequences of radiation damage for water content measurements

References

9 X-ray microanalysis in histochemistry

A The suitability of X-ray microanalysis for histochemistry

B Limitations of X-ray microanalysis in histochemistry

C Fields of application of X-ray microanalysis in histochemistry

1 Tracers

2 Histochemical procedures with colourless or non-electrondense reaction products

3 Validation of histochemical methods

4 Simultaneous localisation of more than one substance:distribution studies

5 Ultrastructural cytochemistry

D Quantification in histochemical X-ray microanalysis

E Specimen preparation for histochemistry

F Concluding remarks

References

10 Sprayed microdroplets: methods and applications

A Introduction

1 Perspective: directly deposited microdroplets

2 Sprayed microdroplets: theoretical background

3 Hybrid techniques: direct deposition with reference elements

B Practical considerations

1 Spraying procedures

2 Analytical throughput

3 Precision of the method

4 Microdroplet stability under electron irradiation

C Applications

1 Standards and calibration

2 The analysis of biological fluids

3 In conjunction with the quantitation of thin sections of biological tissues

4 Analysis of digested, solubilised biological tissues

Acknowledgements

References

11 X-ray microanalysis of biological fluids: applications to investigations in renal physiology

A Introduction

B The microdroplet technique

1 Principle

2 Sample preparation

3 Electron probe analysis procedure

4 Results

(i) LINEARITY OF THE CALIBRATION LINES

(ii) MINIMUM DETECTABLE CONCENTRATION

(iii) MAXIMUM DETECTABLE CONCENTRATION

(iv) PITFALLS

C Applications in renal physiology: renal handling of electrolytes, with special references to magnesium

1 In vitro studies of the effects of ADH, PTH, calcitonin,glucagon and isoproterenol on the transport of electrolytes and Mg in the thick ascending limb

(i) EFFECTS ON SODIUM CHLORIDE TRANSPORT

(ii) EFFECTS ON MAGNESIUM AND CALCIUM TRANSPORT

2 In vivo studies of the hormonal control of the transport of Mg2+ and other related electrolytes

(i) PROXIMAL TUBULE

(ii) LOOP OF HENLE

(iii) DISTAL TUBULE

(iv) HORMONAL CONTROL ON THE RENAL HANDLING OF Mg

Acknowledgements

References

SECTION D APPLICATIONS OF X-RAY MICROANALYSIS IN BIOLOGY

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

B Applications of X-ray microanalysis to different types of biological specimen

References

12 Electron probe X-ray microanalysis of bacterial cells: general applications and specific studies on plant pathogenic bacteria

A Introduction

1 General applications of the technique to bacterial cells

2 Elemental composition of plant pathogenic bacteria

B Experimental use of X-ray microanalysis with plant pathogenic bacteria

1 Specimen preparation and elemental localisation in bacterial cells

(i) CRYOSECTIONS

(ii) WHOLE CELL PREPARATIONS

(iii) EXTRACTED CELL CONSTITUENTS

(iv) ELEMENTAL CORRELATIONS

2 X-ray microanalysis of bacteria cultured in vitro

(i) JJV VITRO CULTURES AS EXPERIMENTAL MODEL SYSTEMS

3 Elemental composition of bacteria in infected plant tissue

(i) COMPARISON WITH BACTERIA CULTURED IN VITRO

(ii) HOST/PATHOGEN INTERACTIONS

C General conclusions and future prospects

References

13 Ion localisation in plant cells using the combined techniques of freeze-substitution and X-ray microanalysis

A X-ray microanalysis of plant cells

B Preparation of freeze-substituted tissue

1 Ion localisation within the halophyte Suaeda maritima

2 Ion distribution in crop plants

3 Ion compartmentation in halophytic microorganisms

C Frozen-hydrated bulk tissue

D Conclusions and the prospects for future research using X-ray microanalysis

References

14 Electron probe X-ray microanalysis of diffusible ions incells and tissues from invertebrate animals

A Introduction

B Measurements of ionic concentration: some technical considerations

C Biological information

1 Intracellular ions

(i) CYTOPLASM

(ii) CELL DIVISION

(iii) SPERM-NUCLEI

(iv) INTRACELLULAR Ca STORES

2 Nematocysts and trichocysts

3 Pericellular compartments

(i) BASEMENT MEMBRANES (BM)

References

15 X-ray microanalysis in pollution studies

A Introduction

B Methodology

C Metals in marine organisms

D Marine pollution effects

Acknowledgements

References

16 X-ray microanalysis in biomaterials research

A Introduction

B The total alloplastic middle ear

C In vitro biodegradation tests

D In vivo biocompatibility tests

E X-ray microanalysis and bone formation

F Summary

References

17 Applications of X-ray microanalysis in biomedicine: an overview

A Summary

B Introduction

C Specimen preparation

D Quantitative analysis

E Applications

1 Cell physiology

2 Cell proliferation and cancer

3 Epithelial ion transport

References

18 X-ray microanalysis of cultured mammalian cells

A Introduction

B Preparation methods

1 Cell culture

2 Removal of medium

3 Fixation

4 Analysis

C Applications

References

Index