Vibrational Spectroscopic Imaging for Biomedical Applications Book

Contributors

Preface

1 Toward Automated Breast Histopathology Using Mid-IR Spectroscopic Imaging R. Bhargava Bhargava, R. 1

1.1 Introduction 2

1.1.1 FT-IR Imaging 4

1.1.2 FT-IR Spectroscopic Characterization of Cells and Tissues 5

1.1.3 FT-IR Imaging for Pathology 6

1.1.4 High-Throughput Sampling 8

1.1.5 Modified Bayesian Classification and Automated Tissue Histopathology 8

1.2 Materials and Methods 10

1.2.1 Models for Spectral Recognition and Analysis of Class Data 11

1.2.2 Automated Metric Selection and Classification Protocol Optimization 12

1.2.3 Spectral Metrics and Biochemical Basis 14

1.2.4 Validation and Dependence on Experimental Parameters 15

1.2.5 Application for Cancer Pixel Segmentation 18

1.2.6 Application for Patient Cancer Segmentation 21

1.3 Conclusions 25

References 26

2 Synchrotrom-Based FTIR Spectromicroscopy and Imaging of Single Algal Cells and Cartilage Carol J. Hirschmugl Hirschmugl, Carol J. 29

2.1 Introduction 30

2.2 IR Environmental Imaging 31

2.2.1 Beamline Design and Implementation 32

2.2.2 Initial Measurements with IRENI 35

2.3 Flow Cell for In Vivo IR Micorspectroscopy of Biological Samples 36

2.3.1 Flow Chamber Design 39

2.3.2 Mid-IR and Vis Measurements 42

2.3.3 Viability Tests: PAM Fluorenscence Measurements 44

2.3.4 Initial Flow Cell Measurements with IRENI 47

2.4 Biomedical Application: Calcium-Containing Crystals in Arthritic Cartilage 48

2.4.1 Calcium-Containing Crystals and Arthritis 48

2.4.2 Current Methods of Crystal Identification 49

2.4.3 Biologic Models of Calcium-Containing Crystal Formation 49

2.4.4 Synchrotron-Based FTIR Microspectroscopy Spectral Analysis of Calcium-Containing Crystals 50

2.5 Future Directions: In Vivo Kinetics of Pathological Mineralization and Phytoplankton Adaptation 55

Acknowledgments 55

References 56

3 Preparation of Tissues and Cells for Infrared and Raman Spectroscopy and Imaging Peter Gardner Gardner, Peter 59

3.1 Introduction 59

3.2 Tissue Preparation 61

3.2.1 Archived Tissue: Paraffin Embedded and Frozen Specimens 61

3.2.2 Preparation of Tissues for Diagnostic Assessment Using FTIR and Raman Microspectroscopy 63

3.2.3 The Effects of Xylene on fixed Tissue and Deparaffinization of Paraffin-embedded Tissue 68

3.3 Cell Preparation 71

3.3.1 Chemical Fixation for FTIR and Raman Imaging 71

3.3.2 Sample Preparation for Biomechanistic Studies 78

3.3.3 Growth Medium and Substrate Effects on Spectroscopic Examination of Cells 80

3.3.4 Preparation of Living Cells for FTIR and Raman Studies 85

3.4 Summary 92

Acknowledgments 94

References 94

4 Evanescent Wave Imaging Andrew P. Evan Evan, Andrew P. 99

4.1 Introduction 99

4.2 Theoretical Considerations 100

4.3 Historical Development 102

4.4 Experimental Implementation 107

4.5 Benefits of ATR Microspectroscopic Imaging for Biological Sections 111

4.6 Macro ATR Imaging 117

4.7 ATR Microspectroscopic Raman Imaging 119

4.8 Conclusions 121

References 121

5 sFTIR, Raman, and SERS Imaging of Fungal Cells Susan G. W. Kaminskyi Kaminskyi, Susan G. W. 125

5.1 Introduction 125

5.2 Introduction to Fungi 127

5.2.1 Specimen Preparation 129

5.3 Vibrational Spectroscopy 130

5.3.1 Spectral Resolution 132

5.3.2 Spatial Resolution 133

5.4 sFTIR Spectra of Fungi 133

5.4.1 Physical Considerations and Spectral Anomalies in sFTIR Spectra 134

5.5 Raman Spectroscopy of Fungi 137

5.5.1 Raman Map From a Hyphe, at Growing Tip 139

5.5.2 Raman Map of Spore Branch 139

5.5.3 Detection of Crystalline Meaterials by IR and Raman 140

5.6 SERS Discovery and Development 142

5.6.1 Substrates: The Key to SERS Imaging 145

5.6.2 SERS: Applications for Fungi 145

5.7 Conclusions: Lessons Learned, Caveats, Challenges, Promise 150

Acknowledgments 151

References 152

6 Widefield Raman Imaging of Cells and Tissues Amy Drauch Drauch, Amy 157

6.1 Introduction 157

6.2 Generation of Raman Images 158

6.2.1 Point Mapping 158

6.2.2 Line Mapping 158

6.2.3 Other Modes of Generating Raman Images 159

6.2.4 Widefield Imaging 159

6.3 Raman Imaging of Cells and Tissues 161

6.4 Background and Image Preprocessing Steps for Widefield Raman Images 164

6.4.1 Fluorescence 164

6.4.2 Correction for Dark Current 166

6.4.3 Cosmic Filtering 166

6.4.4 Instrument Response Correction 166

6.4.5 Flatfielding 167

6.4.6 Baseline Correction 168

6.4.7 Normalization 170

6.4.8 Smoothing 170

6.5 Chemometric Analysis of Widefield Raman Images 170

6.5.1 Principal Component Analysis 171

6.5.2 Mahalanobis and Euclidean Distance 173

6.5.3 Spectral Mixture Resoultion 178

6.5.4 Derivatives 179

6.6 Chemometrics in the Analysis of Non-Widefield Raman Images 180

6.6.1 PCA 181

6.6.2 Linear Discriminant Analysis 184

6.7 Conclusions 187

References 187

7 Resonance Raman Imaging and Quantification of Carotenoid Antioxidants in the Human Retina and Skin Werner Gellermann Gellermann, Werner 193

7.1 Introduction 193

7.2 Optical Properties and Resonance Raman Scattering of Carotenoids 196

7.3 Spatially Integrated Resonance Raman Measurements of Macular Pigment 199

7.4 Spatiallay Resolved Resonance Raman Imaging of Macular Pigment---Methodology and validation Experiments 205

7.5 Spatially Resolved Resonance Raman Imaging of Macular Pigment in Human Subjects 211

7.6 Raman Detection of Carotenoids in Living Human Skin 215

7.7 Conclusions 221

References 222

8 Raman Microscopy for Biomedical Applications: Toward an Efficient Diagnosis of Tissues, Cells, and Bacteria Jurgen Popp Popp, Jurgen 225

8.1 Introduction 226

8.2 Raman Imagining of Tissue 227

8.2.1 Mouse Brains 228

8.2.2 Human Brain Tumors 231

8.2.3 Human colon Tissue 236

8.2.4 Human Lung Tissue 239

8.3 Raman Imaging of Cells 241

8.3.1 Lung Fibroblast cells 242

8.3.2 Red Blood Cells 245

8.4 Raman Spectroscopy of Bacteria 248

8.4.1 Species Classification 248

8.4.2 Imaging Single Bacteria 255

8.5 Conclusions 259

Acknowledgments 260

References 260

9 The Current State of Raman Imaging in Clinical Application Tom C. Bakker Schut Schut, Tom C. Bakker 265

9.1 Introduction 265

9.1.1 History 266

9.1.2 Principles 267

9.2 Instrumentation 268

9.2.1 Laser 269

9.2.2 Microscope 270

9.2.3 Filters 270

9.2.4 Spectrometer 271

9.2.5 CCD 271

9.3 Imaging Techniques 272

9.4 Data Analysis: Spectra to Images(s) 276

9.4.1 Classification Techniques 277

9.4.2 Quantification Techniques 278

9.5 Raman Mapping and Imaging in Bioscience 278

9.5.1 Single Cells 278

9.5.2 Tissues 283

9.6 Limitations and Perspectives 291

References 293

10 Vibrational Spectroscopic Imagning of Microscopic Stress Patterns in Biomedical Materials Giuseppe Pezzotti Pezzotti, Giuseppe 299

10.1 Introduction 300

10.2 Principles of Raman Spectroscopy 302

10.3 Raman Effect in Biological and Synthetic Biomaterials 305

10.3.1 Spectral Features 305

10.3.2 PS Behavior 307

10.4 Visualization of Microscopic Stress Patternsin Biomaterials 309

10.4.1 Micromechanics of Fracture and Crack-Tip Stress Relaxation Mechanisms 309

10.4.2 Residual Stress Patterns on Ceramic-Bearing Surfaces of Artificial Hip Joints 312

10.5 Conclusions 315

References 315

11 Tissue Imaging with Coherent Anti-Stokes Raman Scattering Microscopy Eric Olaf Potma Potma, Eric Olaf 319

11.1 From Spontaneous to Coherent Raman Spectroscopy 319

11.2 The Birth of CARS Microscopy 322

11.2.1 First Generation CARS Microscopes 322

11.2.2 Second Generation CARS Microscopes 323

11.3 CARS Basics 324

11.3.1 Nonlinear Electron Motions 325

11.3.2 Resonant and Nonresonant Contributions 326

11.4 CARS by the Numbers 327

11.4.1 Signal Generation in Focus with Pulsed Excitation 327

11.4.2 Photodamaging 328

11.4.3 CARS Chemical Selectivity 328

11.4.4 CARS Sensitivity 330

11.5 CARS and the Multimodal Microscope 332

11.6 CARS in Tissues 333

11.6.1 Focusing in Tissues 333

11.6.2 Backscattering in Tissues 335

11.6.3 Typical Endogenous Tissue Components 336

11.7 CARS Biomedical Imaging 337

11.7.1 Ex Vivo Nonlinear Imaging 337

11.7.2 In Vivo Nonlinear Imaging 341

11.8 What Lies at the Horizon? 342

Acknowledgments 342

References 342

Index 349