Food microstructures microscopy, measurement and modelling edited by v j morris and k groves

Library of Congress Control Number: 2013944631 ISBN 978-0-85709-525-1 print ISBN 978-0-85709-889-4 online ISSN 2042-8049 Woodhead Publishing Series in Food Science, Technology and

Understanding and controlling the microstructure of complex foods

(ISBN 978-1-84569-151-6)

Designing functional foods: measuring and controlling food structure

breakdown and absorption

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Library of Congress Control Number: 2013944631

ISBN 978-0-85709-525-1 (print)

ISBN 978-0-85709-889-4 (online)

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Contents

Contributor contact details xi

Woodhead Publishing Series in Food Science, Technology and Nutrition xv

Dedication to Brian Hills xxv

Preface xxvii

Introduction xxix

Part I Microstructure and microscopy 1

1 Environmental scanning electron microscopy (ESEM): principles and applications to food microstructures 3

D J Stokes, FEI Company, The Netherlands 1.1 Introduction 3

1.2 Scanning electron microscopy (SEM) 4

1.3 Environmental scanning electron microscopy (ESEM) 7

1.4 Key applications of ESEM for the study of food microstructure 11

1.5 Conclusion and future trends 21

1.6 References 22

2 Probe microscopy and photonic force microscopy: principles and applications to food microstructures 27

V J Morris, Institute of Food Research, UK 2.1 Introduction 27

2.2 Machines and methods: atomic force microscopes 29

2.3 Machines and methods: force spectroscopy 33

2.4 Machines and methods: optical tweezers and photonic microscopy 40

2.5 Applications of the atomic force microscope as a microscope 42

2.6 Applications of atomic force microscopes as a

force transducer 51

2.7 Conclusion 56

2.8 References 57

3 Light microscopy: principles and applications to food microstructures 62

P A Gunning, Smith & Nephew Research Centre, UK 3.1 Introduction 62

3.2 Fundamentals of light microscopy 64

3.3 Specimen preparation 71

3.4 Specimen contrast enhancement: physical methods 77

3.5 Specimen contrast enhancement: chemical and biochemical methods 82

3.6 Interfacial microscopy 90

3.7 Recent and future developments 92

3.8 Conclusion 93

3.9 References 94

4 Confocal microscopy: principles and applications to food microstructures 96

M A E Auty, Teagasc Food Research Centre, Ireland 4.1 Introduction 96

4.2 Principle of confocal microscopy 97

4.3 Chemical contrast: identifying ingredients 100

4.4 Confocal microscopy of food products: a brief review 104

4.5 Model food systems 109

4.6 Refl ectance confocal microscopy 112

4.7 Image processing and analysis 113

4.8 Time dependent studies: dynamic confocal microscopy 115

4.9 Future trends 118

4.10 Conclusion 122

4.11 Sources of further information and advice 122

4.12 References 123

5 Optical coherence tomography (OCT), space- resolved refl ectance spectroscopy (SRS) and time- resolved refl ectance spectroscopy (TRS): principles and applications to food microstructures 132

A Torricelli, Politecnico di Milano, Italy, L Spinelli, IFN-CNR, Italy, M Vanoli, CRA-IAA, Italy, M Leitner and A Nemeth, RECENDT GmbH, Austria and N N D Trong, B Nicolạ and W Saeys, KU Leuven, Belgium 5.1 Introduction 132

5.2 Optical coherence tomography (OCT) 135

5.3 Space- resolved refl ectance spectroscopy (SRS) 142

5.4 Time- resolved refl ectance spectroscopy (TRS) 150

5.5 Conclusion and future trends 156

5.6 Acknowledgements 157

5.7 References 158

6 Fourier transform infrared (FTIR) and Raman microscopy: principles and applications to food microstructures 163

N Wellner, Institute of Food Research, UK 6.1 Introduction 163

6.2 Instrumentation 166

6.3 Data analysis 172

6.4 Applications 177

6.5 Conclusion and future trends 186

6.6 Sources of further information and advice 188

6.7 References 188

7 Ultrasonic and acoustic microscopy: principles and applications to food microstructures 192

M J W Povey and N Watson, Leeds University, UK and N G Parker, Newcastle University, UK 7.1 Introduction 192

7.2 Theories of ultrasound propagation 194

7.3 Construction of an acoustic microscope 204

7.4 Operation and calibration of an acoustic microscope 206

7.5 Exemplars of acoustic microscopy and applications to food structure 214

7.6 Conclusion and future trends 216

7.7 Acknowledgements 220

7.8 References 220

8 Using magnetic resonance to explore food microstructures 223

P S Belton, University of East Anglia, UK 8.1 Introduction 223

8.2 The magnetic resonance experiment 225

8.3 Theoretical background 229

8.4 Practical applications of magnetic resonance systems 236

8.5 Nano- scale magnetic resonance 240

8.6 Conclusion and future trends 241

8.7 Sources of further information and advice 241

8.8 Acknowledgement 242

8.9 References 242

9 X-ray micro- computed tomography for resolving

food microstructures 246

M Barigou and M Douaire, University of Birmingham, UK 9.1 Introduction 246

9.2 Description of X-ray techniques 247

9.3 Theory of X-ray tomography 252

9.4 Contrast, resolution and sample preparation techniques 259

9.5 Applications to food 262

9.6 Conclusion and future trends 266

9.7 References 267

Part II Measurement, analysis and modelling of food microstructures 273

10 Food microstructure and rheology 275

M A Rao, Cornell University, USA 10.1 Introduction 275

10.2 Traditional rheological methods and food structure 275

10.3 Microrheology 284

10.4 Conclusion 289

10.5 References 290

11 Tribology measurement and analysis: applications to food microstructures 292

T B Mills and I T Norton, University of Birmingham, UK 11.1 Introduction 292

11.2 Background tribology 293

11.3 Techniques for measuring tribological parameters 294

11.4 Microstructural infl uences on tribological behaviour 298

11.5 Conclusion and future trends 305

11.6 References 307

12 Methods for modelling food cellular structures and the relationship between microstructure and mechanical and rheological properties 310

S J Cox, Aberystwyth University, UK 12.1 Introduction 310

12.2 Foam structure 311

12.3 Dynamic properties of foams 315

12.4 Rheology 320

12.5 Conclusion 323

12.6 References 323

13 Granular and jammed food materials 325

G C Barker, Institute of Food Research, UK 13.1 Introduction 325

13.2 Packing of granular food material 328

13.3 Jamming in granular materials 331

13.4 Research and developments in the study of granular systems 334

13.5 Conclusion 334

13.6 References 334

14 Modelling and computer simulation of food structures 336

S R Euston, Heriot-Watt University, UK 14.1 Introduction 336

14.2 Molecular simulation methodology 337

14.3 Food biomolecular structure and function: proteins 342

14.4 Food biomolecular structure and function: carbohydrates and triglycerides 350

14.5 Adsorption of food biomolecules 355

14.6 Simulation of food colloids 366

14.7 Conclusion 376

14.8 Acknowledgements 377

14.9 References 377

Appendix: Electron microscopy: principles and applications to food microstructures 386

K Groves, Leatherhead Food Research, UK and M.L Parker, Institute of Food Research, UK A1.1 Introduction 386

A1.2 Techniques and sample preparation 388

A1.3 Applications of electron microscopy (EM) to the understanding of food product structure 404

A1.4 Case studies 411

A1.5 Developments in EM techniques and future prospects 418

A1.6 New challenges and nanotechnology 421

A1.7 Conclusion 422

A1.8 References 423

Index 429

Contributor contact details

(* = main contact)

Editors

Victor J Morris*

Institute of Food Research

Norwich Research Park

Norwich, NR4 7UA, UK E-mail: vic.morris@ifr.ac.uk

Chapter 3

Paul A Gunning Surface Analysis Department Smith & Nephew Research Centre York Science Park

Heslington York, YO10 5DF, UK E-mail: Paul.Gunning@Smith- Nephew.com

Chapter 4

Mark A E Auty Food Chemistry and Technology Department

Teagasc Food Research Centre Moorepark

Fermoy

Co Cork, Ireland E-mail: Mark.auty@teagasc.ie

Chapter 5

Alessandro Torricelli

Dipartimento di Fisica

Politecnico di Milano

Piazza Leonardo da Vinci 32

I-20133 Milan, Italy

E-mail: alessandro.torricelli@polimi.it

Chapter 6

Nikolaus Wellner

Institute of Food Research

Norwich Research Park

Chapter 9

Mostafa Barigou* and Mặlle Douaire School of Chemical Engineering University of Birmingham Edgbaston

Birmingham, B15 2TT, UK E-mail: M.Barigou@bham.ac.uk

Chapter 10

M A Rao Food Process Engineering Department of Food Science Cornell University

Geneva

NY 14456-1447, USA E-mail: mar2@cornell.edu

Chapter 11

T B Mills* and Ian T Norton School of Chemical Engineering University of Birmingham Edgbaston

Birmingham, B15 2TT, UK E-mail: millstb@bham.ac.uk;

I.T.Norton@bham.ac.uk

Institute of Food Research

Norwich Research Park

Mary L Parker Institute of Food Research Norwich Research Park Colney

Norwich, NR4 7UA, UK E-mail: mary.parker@ifr.ac.uk

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239 Encapsulation technologies and delivery systems for food ingredients and

nutraceuticals Edited by N Garti and D J McClements

240 Case studies in food safety and authenticity Edited by J Hoorfar

241 Heat treatment for insect control: Developments and applications D Hammond

242 Advances in aquaculture hatchery technology Edited by G Allan and G Burnell

243 Open innovation in the food and beverage industry Edited by M Garcia

Martinez

244 Trends in packaging of food, beverages and other fast- moving consumer goods

(FMCG) Edited by N Farmer

245 New analytical approaches for verifying the origin of food Edited by P Brereton

246 Microbial production of food ingredients, enzymes and nutraceuticals Edited by

B McNeil, D Archer, I Giavasis and L Harvey

247 Persistent organic pollutants and toxic metals in foods Edited by M Rose and

A Fernandes

248 Cereal grains for the food and beverage industries E Arendt and E Zannini

249 Viruses in food and water: Risks, surveillance and control Edited by N Cook

250 Improving the safety and quality of nuts Edited by L J Harris

251 Metabolomics in food and nutrition Edited by B C Weimer and C Slupsky

252 Food enrichment with omega-3 fatty acids Edited by C Jacobsen, N S Nielsen,

A F Horn and A.-D M Sørensen

253 Instrumental assessment of food sensory quality: A practical guide Edited by

D Kilcast

254 Food microstructures: Microscopy, measurement and modelling Edited by

V J Morris and K Groves

255 Handbook of food powders: Processes and properties Edited by B R Bhandari,

N Bansal, M Zhang and P Schuck

256 Functional ingredients from algae for foods and nutraceuticals Edited by

H Domínguez

257 Satiation, satiety and the control of food intake: Theory and practice Edited by

J E Blundell and F Bellisle

258 Hygiene in food processing: Principles and practice Second edition Edited by

H L M Lelieveld, J Holah and D Napper

259 Advances in microbial food safety Volume 1 Edited by J Sofos

260 Global safety of fresh produce: A handbook of best practice, innovative

commercial solutions and case studies Edited by J Hoorfar

261 Human milk biochemistry and infant formula manufacturing technology

Edited by M Guo

262 High throughput screening for food safety assessment: Biosensor technologies,

hyperspectral imaging and practical applications Edited by A K Bhunia,

M S Kim and C R Taitt

263 Foods, nutrients and food ingredients with authorised EU health claims

Edited by M J Sadler

264 Handbook of food allergen detection and control Edited by S Flanagan

265 Advances in fermented foods and beverages: Improving quality, technologies

and health benefi ts Edited by W Holzapfel

266 Metabolomics as a tool in nutritional research Edited by J.-L Sebedio and

L Brennan

267 Dietary supplements: Safety, effi cacy and quality Edited by K Berginc and

S Kreft

268 Grapevine breeding programs for the wine industry: Traditional and molecular

technologies Edited by A G Reynolds

269 Handbook of natural antimicrobials for food safety and quality Edited by

M Taylor

270 Managing and preventing obesity: Behavioural factors and dietary

interventions Edited by T Gill

271 Electron beam pasteurization and complementary food processing technologies

Edited by S Pillai and S Shayanfar

272 Advances in food and beverage labelling: Information and regulations Edited by

P Berryman

273 Flavour development, analysis and perception in food and beverages Edited by

J K Parker, S Elmore and L Methven

274 Rapid sensory profi ling techniques and related methods: Applications in new

product development and consumer research Edited by J Delarue, B Lawlor and

M Rogeaux

275 Advances in microbial food safety: Volume 2 Edited by J Sofos

276 Handbook of antioxidants in food preservation Edited by F Shahidi

277 Lockhart and Wiseman’s crop husbandry including grassland: Ninth edition

H J S Finch, A M Samuel and G P F Lane

278 Global legislation for food contact materials: Processing, storage and packaging

Edited by J S Baughan

279 Colour additives for food and beverages: Development, safety and applications

Edited by M Scotter

Dedication to Brian Hills

11 June 1949–29 October 2012

I fi rst met Brian when he joined the Institute of Food Research (IFR) to help develop the use of NMR in food science Brian was an undergraduate and postgraduate at Oxford He also did postdoctoral research at MIT and Cambridge prior to joining IFR in 1987 At IFR he became a key research leader whose work was recognised internationally From the time he joined IFR, it was clear that Brian was very knowledgeable, highly motivated and extremely innovative He had a ’hands- on’ approach to science, the ability to identify key problems and to devise novel solutions at both the theoretical and practical level His models for molecular transport in complex media underpin research on food processing, the physical, chemical and microbial stability of foods, the modelling of fl avour encapsulation and release, and the structure and structural changes in cellular materials such as starch, emulsions or plant tissue In terms of NMR applications Brian developed novel pulse sequences to interrogate materials, new theoretical models to interpret the data, and experimented with new types of spectrometers, such as fi eld cycling NMR Recently he invested considerable effort into the use

of his knowledge of water in foods, to develop novel imaging methods directed towards industrial challenges requiring high throughput, low- fi eld, low cost methods, that can be used as sensors in real industrial environments Just prior to his death he had been working on new advances in the acquisition and interpretation

of MRI images, which substantially reduced the acquisition times and enhanced the quality of the images: research of value in the food area but also with wider clinical applications

Brian published alone and with numerous co- authors over 100 peer- reviewed

articles on NMR and its applications, plus standard textbooks such as Magnetic

Resonance Imaging in Food Science and Advances in Magnetic Resonance in Food Science , together with numerous book chapters on NMR methods and

applications Brian was deeply religious and saw physics as a way of glimpsing what he regarded as the wonders of God’s creation His interests in physics were widespread and knowledgeable and he published in other areas outside NMR and food science This broader aspect of his interests is perhaps illustrated by his book

Origins: cosmology, evolution and creation

It was a pleasure to know and work with Brian His ‘hands- on’ approach made him well respected and liked by co- workers He did not suffer fools gladly but, in all the time I knew him, I never heard him say a bad word about anyone Sadly he left us before some of the ideas which he spawned and nurtured were able to blossom, as I hope they will do in the future Brian was to have written a chapter

on NMR and MRI applications in food science Because of his illness this proved impossible I wish to thank Peter Belton for taking on this task at the last minute

I hope this book conveys some of the interest and enthusiasm Brian had for understanding food structure and might inspire others to continue in this area of research

V J Morris, IFR, 2013

Preface

The knowledge of food structure has advanced considerably over the last

30 years Aside from the academic interest in understanding the complex structure

of foods, these advances are enabling the design of new foods to improve their safety and quality, and to enhance the nutritional and health benefi ts of natural and processed foods This improvement in our understanding has resulted from the continued development of new methods for visualising and modelling food microstructure

This book is not intended to provide a detailed description of all the wide and varied types of food structures Rather the intention is to introduce the methodologies available to probe food microstructure and to indicate the type of information that can be obtained through their use By choice the focus is on microscopy and modelling techniques that yield direct information on structure, ranging over different hierarchical levels from the molecular to the macroscopic The level of coverage in different chapters varies, depending on the maturity of the techniques under discussion In all cases, the hope is that suffi cient information

is provided to indicate how the technique works, the type of information obtainable, and the advantages and disadvantages of this method

The literature on food microstructure is vast and continues to expand rapidly The coverage in the chapters is not meant to be exhaustive but rather to emphasise particular points or to provide a route to the literature in particular areas The choice is not meant to indicate priority and omissions are simply a result of the restrictions on space rather than any refl ection on the quality of the publications What do we wish to achieve in editing this book? The aim is to introduce the methods available for visualising and modelling food structure In general terms, the intention is to convey the sort of information that has been obtained, to indicate the progress being made at the present time, and to speculate on what can be achieved in the future Who do we feel should benefi t from reading this book? We hope the book will be of interest to researchers and participants in industry,

research institutes and universities, and to those with major interests not just in food structure but also in the application and design of natural microstructures for pharmaceutical, nutritional or health benefi ts It is hoped that this book will be useful for researchers interested in developing microscopic and modelling techniques, and that it might foster greater collaboration between these two schools, particularly in the food area

We wish to thank the contributors for their participation and for their patience

in awaiting completion of this volume Both of us wish to acknowledge the

fi nancial support from our respective organisations and the guidance and patience

of the editors at Woodhead Publishing during the construction of this volume

V J Morris and K Groves

Introduction

This book is about the methods available to allow food scientists to monitor, visualize or simulate food structures, and consequently understand the changes that occur to such food structures during the cooking, processing or digestion of natural or processed foods

Food structures are complex: they range from the intricate self- assembled structures present in plant and animal tissue to the prefabricated structures generated in processed foods Food structures change with time Most people will

be familiar with the softening of strawberries or tomatoes on ripening, or the loss

of the crunchiness of pears and apples on storage, associated with the gradual breakdown of their structures All processed foods have fi nite shelf lives, often associated with the deterioration of their internal structure In the current climate

of concerns over health, obesity, environmental issues, waste and food shortages, the shelf- life and quality of processed foods is of key importance Understanding how the ingredients make up the structure of the foods and how this structure changes during its life or on eating will play an important part in the development and management of the food industry

Observing how foods fl ow or deform when subjected to stress can monitor these structural changes The techniques used to study these properties are called mechanical and rheological methods and these methods are discussed in this book The instruments range from simple, cheap empirical methods, through comparatively cheap quality control instruments, to sophisticated, expensive machines for detailed characterization of food systems In addition to defi ning structure and structural changes, the techniques can be used to extract parameters that refl ect textural changes, and can be related to certain perceived sensory attributes of food However, this latter aspect will not be considered in the present book

The behaviour of food systems changes when they are confi ned to small regions between surfaces The study of such behaviour requires specialized

equipment and these techniques are called tribology: this is a new, emerging fi eld

of study which is important for studying structural changes in the mouth or during complex processing operations

Although not new, there is a growing interest and development in methods for using probes to monitor the internal structure of food systems These micro- rheological methods observe the constrained oscillation or restricted meandering

of the probes through the food structure In their earliest incarnation, these methods were largely restricted to studies on transparent food systems, but nowadays can be used increasingly to investigate complex opaque food systems, and their use will be described and discussed

Apart from simple studies on dilute molecular solutions, rheological techniques cannot provide direct information on the underlying food structure, at least at the molecular level The interpretation of the rheological and mechanical properties relies on structural information derived mainly from microscopic and imaging methods and, increasingly, on the use of computer simulations to test models for food structure

The development of the fi rst (optical) microscope literally opened a window with which to visualize and describe the structure of materials New microscopic techniques have proved equally signifi cant and nowadays the food scientist has a wide variety of microscopic methods, which are available to probe food structure

at different hierarchical levels Some of these methods are extremely well established but are still benefi ting from new instrumental developments, others are still emerging, and some are still in their infancy, at least in their use in food science The use of new microscopic methods to study foods has revealed additional structural information and new insights and applications in food science and technology

The major structural components of natural and processed foods are biological molecules: carbohydrates, proteins and lipids (fats) These components can self- assemble, or be induced to assemble into higher- order structures, and these structures themselves can be components of even more complex food structures Examples of such structures include the self- assembly of proteins to form the

fi brous structure of meat, or the self- assembly of polysaccharides to form the granule structure of starch or the cellular network structures of plant cell walls Association of globular proteins and polysaccharides can be used to create fi brous

or particulate gels and the assembly of fats (surfactants) or proteins at interfaces can be used to prepare and stabilize foams and emulsions

Processing can be used to develop more complex structures in foods such as ice cream, which is a solid milk fat emulsion containing air bubbles, ice, sugar and fat crystals, or the aerated structures of cakes and bread Thus a range of microscopic and imaging techniques are required to span the different structural regimes that can be present in food materials The intention of this book is to introduce the range of microscopic and imaging methods currently available to investigate such food structures

Optical microscopy is the oldest, most established and most versatile method for studying food materials As early as 1665, Robert Hooke reported images of

plant materials in his book ‘Micrographia’ and introduced the concept of biological cells The advantages of optical microscopy are the ease of use, relatively low cost, and the wide variety of contrast mechanisms and stains The development of the phase contrast microscope alone was suffi ciently signifi cant to justify the award of the Nobel Prize in Physics to Fritz Zernike in 1953 Extension of the operating range into the infra- red, through the use of Infra- red microscopy (developed fi rst in the mid-1950s) and Raman microscopy (developed in the mid-1970s) has allowed the mapping of different structural components, together with additional information on their physical state within complex food structures Finally, the arrival of confocal laser scanning microscopy in the late 1980s rejuvenated the use of optical techniques, particularly in the use of the methods to follow processing operations The resolution achievable with optical and infra- red methods is limited by the wavelength of the radiation used to probe the sample Thus, although the techniques can identify the presence of different molecular species, it is not possible to image the structures at molecular resolution

Ernst Ruska constructed the fi rst electron microscope in 1933, although he and Maximillion Knoll obtained the fi rst images earlier, in 1931 Physicists and material scientists were the main users of the technique until 1959, when R (Bob)

W Horne and Sydney Brenner developed the technique of negative staining, opening up the use of the technique for biologists The use of transmission electron microscopy (TEM) allowed the molecular structure of foods to be probed for the

fi rst time TEM and the companion technique of scanning electron microscopy (SEM, fi rst marketed in the mid-1960s) require that the samples are imaged under vacuum, and this has led to the development of elegant preparative methods to preserve the ‘native’ structure of food samples The development of the environmental scanning electron microscope and similar ‘low vacuum’ SEMs (the ESEM became available in the late 1980s) is now allowing the imaging of

‘wet’ food samples, although the resolution achievable still lags behind that obtainable by conventional SEM This remains a new and developing technique Gerd Binnig and Heinrich Rohrer developed the scanning tunnelling microscope (STM) in the early 1980s, a discovery that won them the Nobel Prize

in Physics in 1986, together with Ernst Ruska for the development of the electron microscope This discovery of the STM led to the development of a family of microscopes (probe microscopes), which image by feeling, rather than visualizing

a surface The most versatile member of this family for studying biological samples is the atomic force microscope (AFM), developed by Binning and colleagues in 1986 and fi rst commercialized in the early 1990s Applications in food science began in that period and have expanded through the development of successive generations of AFMs

A related technique that can generate images by feeling is that of optical tweezers: a laser beam is used to trap a probe particle and monitor its interaction with the internal structure of complex materials This can be used to generate a 3D map of the structure and these microscopes are called photonic microscopes The development of this technique in food science has been restricted by the lack of commercial instruments that are now only starting to become available The

ability of the AFM and optical tweezers to measure the forces between derivatized probes and samples allows them to determine and map the forces involved in the assembly of food structures at the molecular and colloidal level These techniques

of molecular and colloidal force spectroscopy are new and emerging techniques

in food science

A number of non- invasive imaging techniques have been developed principally for clinical use Examples include acoustic microscopy (introduced in the mid-1970s), optical coherence tomography (developed in the late 1980s to early 1990s) and the related techniques such as space- or time- resolved refl ectance spectroscopy, X-ray micro- computed tomography (also introduced in the early 1970s) and magnetic resonance imaging (MRI)

Of these methods, perhaps the most used in food science is MRI The construction of the fi rst MRI machine is attributed to Raymond Damadian in

1971, with the fi rst MRI image obtained by Paul Christian Lauterbur in 1973 Sir Peter Mansfi eld developed mathematical procedures and techniques for enhancing the clarity and acquisition of images The signifi cance of the clinical applications

of MRI led to the award of the Nobel Prize in Physiology or Medicine to Lauterbur and Mansfi eld in 2005 Applications of NMR and MRI for studying food structure began in the early 1970s and have expanded since that time The intention in this book is to focus on the investigation of food structure, and also the changes in food structure within the body on digestion, rather than the clinical uses for mapping the consequent fat distribution in the body, or changes in brain activity associated with food consumption

A new and emerging area of research on food structure has been made possible

by the dramatic improvement in computing power and speed This has led to the use of computer simulations, which can be used to run and examine models for food structure This area is covered by a description of modelling and simulation techniques and their applications to food systems, plus descriptions of the modelling of particular generic food structures, which include granular materials and cellular structures in foods These methods provide a link between the microscopic elements of food structure and the nature and behavior at the macroscopic level

What do we wish to achieve in writing this book? One aim is to collect together the large and increasing number of methods available to characterize food structure and the changes that occur on the formation and breakdown of such structures The coverage of different methods is variable, depending on how new

or established these methods are in investigating food structures However, the hope is that the coverage is suffi cient in all cases to introduce the methods, to demonstrate the types of information that can be obtained, and the types of structures that can be studied In general terms, the intention is to look at what can

be done, how it is done and where things may go in the future We hope the book will provide a good resource base for the literature on techniques for probing food structure and provide a basis for understanding how such techniques have been used to characterize food structure For the well- established methods, mainly microscopies, which have contributed to our present views on food structure, the

intention is to describe how they can now be used routinely (at least in the hands

of experts) to characterize foods and food structures In the case of new and emerging techniques, the hope is that the reader will get a feel for the new insights these methods are providing, and could provide in the future

During both of our careers, the methods available to study food structure and the new insights and understanding of complex foods they have provided have increased dramatically We feel this is likely to continue in the future We hope this book provides a picture of the current state of the art and a springboard to future developments It is always diffi cult and perhaps foolish to try to predict the future The discovery and development of probe microscopes is clearly an example

of a technique which could not have been predicted in advance However, certain aspects of the study of food structure are predictable It is clear that the development

of hybrid instruments combining different forms of microscopy will continue, extending the range and nature of images of food structure

At present, the resolution achievable with most microscopic techniques is limited by the wavelength of the incident radiation This limitation can be overcome through the use of near- fi eld methods, where the source is brought to within less than the wavelength from the surface At present, the signal- to-noise ratios for such methods are generally low, and the acquisition times are long It is

to be expected that these largely technological problems will be reduced in the future, opening up the use of these methods Computing power and availability is likely to continue to increase This will probably improve the speed of acquisition, processing and presentation of images We might expect the use of modelling and simulations to become more routine and widely used in food science The availability of high power sources of radiation, such as synchrotrons, means that the ability to model kinetics is likely to be complemented by new experimental data

Nowadays there is an increasing demand for functional foods designed rationally to enhance health and reduce the risks of contracting long- term chronic diseases Developing such products requires the construction of foods that are acceptable to the consumer in terms of cost, taste, texture and appearance In order

to deliver health benefi ts it is necessary to tailor the breakdown of the structure during digestion to facilitate release of structural components, and to optimize uptake and transport within the body The key to success is to understand how to design and construct the correct food matrix

This book covers the methods available for probing or simulating the assembly and stability of food structure, and for selecting and monitoring the site and mode

of breakdown during digestion Thus we hope that it will be of interest to students and researchers interested in food structure and to food scientists and technologists faced with the continuous and growing demand for the production of safer and healthier functional foods

V J Morris and K Groves

1

Environmental scanning electron

microscopy (ESEM): principles and

applications to food microstructures

D J Stokes, FEI Company, The Netherlands

DOI: 10.1533/9780857098894.1.3

Abstract: This chapter introduces some basic principles of scanning electron microscopy

(SEM) and its extension to environmental scanning electron microscopy (ESEM), describing why ESEM is useful for characterising materials of interest in food research

It fi rst surveys the main techniques of imaging and microanalysis in SEM The principles

of ESEM are then described, explaining how gases can be used to mitigate electrical charging of uncoated insulating materials and contribute to the image formation process, and other ways in which gases and specimen temperature control are useful for

expanding the range of available techniques to yield additional information about

the structure–property relations of hard, soft and even liquid specimens Several

key application techniques will be covered, from general imaging of dry or moist,

uncoated specimens through to in situ dynamic experiments

Key words: scanning electron microscopy (SEM), environmental scanning electron

microscopy (ESEM), gases, aqueous and hydrated specimens, in situ , dynamic

experiments, cryoESEM

1.1 Introduction

The main themes of this chapter are scanning electron microscopy (SEM) and its extension to environmental scanning electron microscopy (ESEM), with examples demonstrating the ways in which ESEM can be used for characterising materials

of interest in food research These materials range from confectionery and cereal products to fl uid- fi lled vegetable cells and tissues through to emulsions, highlighting the diversity of material types that can be accommodated in the ESEM without the need for extensive specimen preparation traditionally associated with high vacuum electron microscopy

Section 1.2 surveys the basic components of the SEM, the requirements for placing specimens in high vacuum and the main techniques of imaging and microanalysis The principles of ESEM are then described in Section 1.3, explaining how gases can be used to mitigate electrical charging of uncoated, insulating materials and contribute to the image formation process The section also explains other ways in which gases and specimen temperature control are useful for expanding the range of available techniques to yield additional information about the structure–property relationships of hard, soft and even liquid specimens Several key application techniques are covered in Section 1.4, from general imaging of uncoated specimens, through to in situ dynamic

experiments such as wetting and drying, mechanical testing and freezing The chapter concludes with Section 1.5, briefl y discussing the outlook for ESEM in the study of food microstructure

1.2 Scanning electron microscopy (SEM)

SEM has its beginnings in the 1930s (Knoll, 1935; von Ardenne, 1938a,b) and

continued its development through the 1940s onwards (Zworykin et al , 1942;

McMullen, 1953; Smith and Oatley, 1955; Everhart and Thornley, 1960), becoming commercially available in 1965 With it came the ability to study the microstructural characteristics of bulk materials with large depth of fi eld across length scales ranging from millimetres to nanometres, offering a valuable new addition to the suite of visual characterisation tools such as light microscopy and scanning/transmission electron microscopy (S/TEM)

Typically, an SEM consists of an electron source to generate a beam of primary electrons; a column with electromagnetic lenses for focusing and demagnifying the primary electron beam; coils for scanning the electron beam across the specimen surface; a chamber containing a stage to hold the specimen; vacuum pumps to maintain the system under high vacuum (usually of the order of

10 −5 –10 −7 Pa); and one or more detectors for collecting signals generated

by electron irradiation of the specimen Finally, the magnifi ed image is displayed on a monitor, as the beam is scanned pixel- by-pixel across the fi eld- of-view

The most straightforward specimen types for SEM are metals, primarily since these materials are less prone to the effects of charging and damage under electron irradiation in high vacuum Methods have evolved to address the issue of imaging electrically insulating materials such as polymers and ceramics, including coating the surface with conductive materials; incorporating heavy metal salts into the specimen to increase bulk conductivity, especially for biological materials; or using low voltages to minimise the accumulation of negative charge within the specimen, making it possible to image uncoated materials (Goldstein, 2003) For materials classes that are not naturally solids or have a tendency to outgas in vacuum, there are methods for conferring rigidity and preventing outgassing so that the specimen is suitable for high vacuum conditions in the SEM These

include critical point drying, freeze drying and the use of cryo- stages for frozen- hydrated specimens (cryoSEM)

Section 1.2.1 gives a brief overview of imaging in the SEM, mainly concentrating on beam–specimen interactions For further reading on the topic as

a whole, the interested reader is referred to Reimer (1985), Newbury et al (1986), Sawyer and Grubb (1987), Goodhew et al (2001) and Goldstein et al (2003)

in Fig 1.1

BSEs are generated via elastic (non energy- absorbing) scattering of primary electrons within the specimen and are defi ned by convention as having energies from 50 eV all the way up to the primary electron energy of the source, which is usually in the range 1 to 30 keV BSEs are thus essentially primary electrons that

Fig 1.1 Diagram showing a range of signals in the SEM For bulk specimens, these

include backscattered and secondary electrons, various photons such as X-rays and visible light and Auger electrons For thin specimens, transmitted electrons provide information

from the degree and nature of scattering

re- emerge from the specimen surface after a series of trajectory- altering interactions with the Coulombic fi eld around atoms in the specimen Inelastic energy loss mechanisms also come into play, causing electrons to transfer energy

to the atoms of the specimen; hence BSEs are emitted from the surface with a range of energies (or are absorbed by the specimen) BSEs can travel from comparatively large depths (10 2 –10 3 nm) to reach the surface, and the BSE emission co- effi cient increases as a function of atomic number, so the BSE signal generally gives rise to images that refl ect compositional information rather than being surface- sensitive

SEs, of which there are several types, are produced via inelastic interactions with primary electrons Here, the generated electrons (known as type I, or SE I ) originate from the specimen’s atoms as primary electrons excite atomic orbital electrons suffi cient to result in ionisation SEs are defi ned as having energies up

to 50 eV, but are typically emitted at around a few eV Other types of SE include

SE II , generated by BSEs as they exit the specimen surface and interact with atoms

as they pass by, and SE III , generated by primary electrons striking the polepiece and chamber walls The low- energy nature of SEs means that the signal is emitted from very close to the surface (< 50 nm) – SEs are prone to inelastic, energy- absorbing processes and so have a short escape depth – giving rise to images that are characteristically topographic, showing surface relief SE imaging thus provides complementary information to the BSE signal SE signals are sensitive

to variations in primary electron beam energy, since this affects the depth at which SEs are produced With increasing beam energy, primary electrons penetrate further into the material, particularly in soft materials such as polymers, resulting

in a lower SE yield, compared to lower beam energies where SEs are generated closer to the surface and so a greater proportion can escape As a general rule, SE imaging of fi ne surface features is best carried out at lower beam energies Inelastic scattering also produces characteristic and continuum (Bremsstralung) X-rays, Auger electrons, electron- hole pairs (excitons), long- wavelength electromagnetic radiation (cathodoluminescence), lattice vibrations (phonons) and collective electron oscillations (plasmons) X-ray photons are often used for microanalysis in the SEM, the most common form of detection being energy dispersive spectroscopy (EDS), and are produced as a result of the relaxation of

an excited state in the atom following primary electron irradiation X-ray energies are thus directly related to the chemical elements in the specimen, thus providing quantitative or qualitative information about chemical composition, either via spectra or mapping of elements present

In the absence of a conductive coating, electrically insulating materials are especially susceptible to both radiation damage and electrical charging For example, organic materials can become noticeably damaged during electron excitation and ionisation, and atoms can become displaced, changing the structure and appearance visible at the surface Other effects include breaking of bonds, cross- linking, mass loss (e.g through the formation and liberation of volatile components), temperature change and the formation of a carbonaceous deposit (contamination) Care must be taken to minimise these effects by careful choice

of electron beam energy and fl ux, specimen thickness and temperature More detailed information can be found in Talmon (1987)

Electrical charging is a particular problem: electrons accumulate in the specimen, leading to electric fi elds that distort or defl ect the primary electron beam This can signifi cantly interfere with the ability to interpret images, or even

to obtain an image at all Again, appropriate choice of beam energy, fl ux and specimen thickness can be employed to help mitigate the problem (Joy and Joy,

1998; Goldstein et al , 2003), but there are certain inter- dependencies that make it

diffi cult to satisfy the conditions needed to control both radiation damage and charge build- up at the same time, particularly for heterogeneous specimens Hence, together, these are the main reasons why conductive coatings are applied

to electrically insulating materials, especially those of a delicate, organic nature However, such specimen preparation, including the steps necessary to render the specimen ready for coating, can involve lengthy procedures that may introduce changes to or obscure features of interest

1.3 Environmental scanning electron microscopy (ESEM)

In order to ease some of the sample preparation and handling requirements for SEM noted above and so allow a wider range of observation conditions, particularly for insulating and non- solid materials, the SEM was adapted for greater fl exibility This involved the introduction of gases into the specimen area

In essence, the use of a gas serves the purposes of mitigating charging effects in insulators, providing an alternative mechanism for electron signal amplifi cation, and enabling hydrated/liquid specimens to be observed directly in their natural state These attributes make it possible to eliminate many of the specimen preparation steps that are sometimes required in the SEM, enabling the natural surfaces of delicate or otherwise challenging materials to be observed without a

conductive coating and allowing for dynamic in situ experiments, as well as

helping to avoid artefacts that can arise as a result of freezing and/or drying ordinarily required to render specimens solid for high vacuum SEM imaging Early demonstrations of such capabilities include Lane (1970), Robinson (1974, 1975), Danilatos and Robinson (1979) and Shah and Beckett (1979) First commercialised around 1980, the technology became more widely available in the 1990s, bringing yet further insights into the structure–property relationships

of many materials that were previously unsuitable for study without preparation for conventional SEM

Given that materials of relevance to food research are predominantly electrically insulating in physical character and are sometimes hydrated or liquid in their natural state, the various derivatives of this type of SEM have a clear place amongst the tools used for the study of food structure and properties The technology is popularly known by terms such as environmental SEM (ESEM) and variable pressure SEM (VPSEM), amongst others For a description of these terms, see Stokes (2008) ESEM is one of the more well- known versions of the