Preface ..................................................... XV
List of Contributors ...................................... XVII
Part One Basic Methodology
1 Infrared and Raman Instrumentation for Mapping and
Imaging ...................................................... 3
Peter R. Griffiths
1.1 Introduction to Mapping and Imaging ..................... 3
1.2 Mid-Infrared Microspectroscopy and Mapping .............. 5
1.2.1 Microscopes and Sampling Techniques .............. 5
1.2.2 Detectors for Mid-Infrared Microspectroscopy ..... 9
1.2.3 Sources for Mid-Infrared Microspectroscopy ...... 12
1.2.4 Spatial Resolution .............................. 15
1.2.5 Transmission Microspectroscopy .................. 20
1.2.6 Attenuated Total Reflection Microspectroscopy ... 21
1.3 Raman Microspectroscopy and Mapping .................... 24
1.3.1 Introduction to Raman Microspectroscopy ......... 24
1.3.2 CCD Detectors ................................... 29
1.3.3 Spatial Resolution .............................. 31
1.3.4 Tip-Enhanced Raman Spectroscopy ................. 33
1.4 Near-Infrared Hyperspectral Imaging .................... 36
1.5 Raman Hyperspectral Imaging ............................ 41
1.6 Mid-Infrared Hyperspectral Imaging ..................... 45
1.6.1 Spectrometers Based on Two-Dimensional Array
Detectors ....................................... 45
1.6.2 Spectrometers Based on Hybrid Linear Array
Detectors ....................................... 50
1.6.3 Sampling ........................................ 52
1.7 Mapping with Pulsed Terahertz Radiation ................ 56
1.8 Summary ................................................ 60
Acknowledgments ............................................. 62
References .................................................. 62
2 Chemometric Tools for Image Analysis ........................ 65
Anna de Juan, Marcel Maeder, Thomas Hancewicz,
Ludovic Duponchel, and Romà Tauler
2.1 Introduction ........................................... 65
2.2 Hyperspectral Images: The Measurement .................. 66
2.2.1 The Data Set and the Underlying Model ........... 66
2.3 Image Preprocessing .................................... 68
2.3.1 Signal Preprocessing ............................ 69
2.3.1.1 De-noising ............................. 69
2.3.1.2 Baseline Correction .................... 69
2.3.1.3 Detection and Suppression of
Anomalous Pixels or Anomalous
Spectral Readings ...................... 70
2.3.2 Data Pretreatments .............................. 70
2.3.3 Image Compression ............................... 71
2.4 Exploratory Image Analysis ............................. 72
2.4.1 Classical Image Representations ................. 72
2.4.2 Multivariate Image Analysis (MIA) and
Principal Component Analysis (PCA) .............. 72
2.5 Quantitative Image Information: Multivariate Image
Regression (MIR) ....................................... 77
2.6 Image Segmentation ..................................... 80
2.6.1 Unsupervised and Supervised Segmentation
Methods ......................................... 80
2.6.2 Hard and Fuzzy Segmentation Approaches .......... 83
2.7 Image Resolution ....................................... 84
2.7.1 The Image Resolution Concept: Monolayer and
Multilayer Image Analysis ....................... 84
2.7.2 Spatial and Spectral Exploration ................ 85
2.7.3 The Resolution Process: Initial Estimates and
Constraints ..................................... 89
2.7.4 Resolution on Compressed Images ................. 95
2.7.5 Resolution and Available Library Spectra ........ 97
2.7.6 Multilayer and Multi-Image Resolution .......... 200
References ................................................. 106
Part Two Biomedical Applications
3 Vibrational Spectroscopic Imaging of Soft Tissue ........... 113
Christoph Krafft
3.1 Introduction .......................................... 113
3.1.1 Epithelium ..................................... 114
3.1.2 Connective Tissue and Extracellular Matrix ..... 115
3.1.3 Muscle Tissue .................................. 116
3.1.4 Nervous Tissue ................................. 117
3.2 Preparation of Soft Tissue for Vibrational
Spectroscopic Imaging ................................. 118
3.2.1 General Preparation Strategies ................. 118
3.2.2 Vibrational Spectra of Reference Materials ..... 119
3.2.1 Preparation for FT-IR Imaging .................. 121
3.2.4 Preparation for Raman Imaging .................. 123
3.3 Applications to Soft Tissues .......................... 124
3.3.1 Colon Tissue ................................... 124
3.3.2 Brain Tissue and Brain Tumors .................. 128
3.3.2.1 Whole Mouse Brains .................... 129
3.3.2.2 Primary Brain Tumors .................. 131
3.3.2.3 Secondary Brain Tumors ................ 133
3.3.3 Cervix Uteri and Squamous Cell Carcinoma ....... 135
3.3.4 Skin ........................................... 139
3.3.4.1 Corneocyte Biology .................... 139
3.3.4.2 Pharmacology .......................... 140
3.3.4.3 Disease and Cancer Diagnosis .......... 140
3.3.4.4 Raman Systems for Skin Studies ........ 141
3.3.5 Ocular Tissue .................................. 141
3.3.5.1 Macular Degeneration of the Retina .... 142
3.3.5.2 Chemical Composition of the Vitreous
and the Lens .......................... 142
3.4 Conclusions ........................................... 142
References ............................................ 143
4 Vibrational Spectroscopic Imaging of Hard Tissues .......... 149
Michael D. Morris, Matthew V. Schulmerich,
Kathryn A. Dooley, and Karen A. Esmonde-White
4.1 Infrared and Raman Spectroscopy of Bone ............... 149
4.2 Infrared and Raman Imaging of Bone .................... 150
4.2.1 Introduction ................................... 150
4.2.2 Imaging of Normal, Healthy Mineralized Tissue
as a Function of Age ........................... 151
4.2.3 Adaptation of Bone Induced by Genetic
Modifications .................................. 154
4.2.4 Adaptation of Bone in Response to External
Stress ......................................... 156
4.2.5 Adaptation of Bone in Response to
Osteoporosis ................................... 159
4.3 Infrared and Raman Spectroscopy of Cartilage .......... 162
4.4 Infrared and Raman Imaging of Cartilage ............... 164
4.4.1 Unmineralized Cartilage Tissue ................. 164
4.4.2 Mineralized Cartilage and Subchondral Bone ..... 165
4.4.3 Applications in Tissue Engineering ............. 166
4.5 Conclusions ........................................... 167
References ............................................ 168
5 Infrared and Raman Spectroscopy and Spectral Imaging of
Individual Cells ........................................... 173
Max Diem, Christian Matthäus, Tatyana Chernenko,
Melissa J. Romeo, Miloš Miljković, Benjamin Bird,
Jennifer Schubert, Kostas Papamarkakis, and Nora Laver
5.1 Introduction .......................................... 173
5.2 Methods ............................................... 175
5.2.1 Cell Collection and Culturing Methods .......... 175
5.2.1.1 Exfoliated Cells ...................... 175
5.2.1.2 Cultured Cells ........................ 176
5.2.2 Sample Preparation ............................. 177
5.2.2.1 Sample Substrates ..................... 177
5.2.2.2 Fixation .............................. 177
5.2.2.3 Sample Deposition ..................... 177
5.2.3 Data Acquisition ............................... 178
5.2.3.1 Raman Spectroscopy and Raman
Spectral Mapping ...................... 178
5.2.3.2 Infrared Instrumentation .............. 179
5.2.4 Methods of Data Analysis ....................... 179
5.2.4.1 Data Pre-Processing ................... 179
5.2.4.2 Principal Component Analysis (PCA) .... 180
5.2.4.3 Hierarchical Cluster Analysis (HCA) ... 181
5.3 Results and Discussion ................................ 182
5.3.1 General Features of Infrared and Raman
Spectra of Cells ............................... 182
5.3.2 Infrared Cytology .............................. 184
5.3.2.1 Oral Mucosa Cells ..................... 184
5.3.2.2 Human Urine-Borne Cells ............... 185
5.3.2.3 Human and Canine Cervical Cells ....... 188
5.3.3 Results from Confocal Raman Microspectroscopy
of Human Cells ................................. 192
5.3.3.1 Morphology and Subcellular
Organization of Cells ................. 192
5.3.3.2 Visualization of Mitosis .............. 196
5.3.3.3 Liposome Uptake into Cells ............ 196
5.4 Conclusions ........................................... 199
Acknowledgments ....................................... 199
References ............................................ 199
6 3-D Imaging of Biomedical Samples .......................... 203
Don McNaughton, Bayden R. Wood, Timothy С. Сох,
Jörg-Detlef Drenckhahn, and Keith R. Bambery
6.1 Introduction .......................................... 203
6.2 Methodologies ......................................... 204
6.2.1 Sample Preparation and Spectroscopy ............ 204
6.2.2 Univariate and Multivariate Image
Construction ................................... 205
6.2.3 Artificial Neural Network Image Construction ... 208
6.3 Resultant 3-D Images .................................. 209
6.3.1 Chemimage and UHCA Stack Plots ................. 209
6.3.2 3-D Chemimage and UHCA Images .................. 210
6.3.2.1 Cervical Adenocarcinoma ............... 210
6.3.2.2 Adult Mouse Heart ..................... 215
6.3.2.3 3-D ANN Images ........................ 217
6.4 Conclusions ........................................... 219
Acknowledgments ............................................ 220
References ................................................. 220
Part Three Agricultural Applications
7 FT-IR Microspectroscopic Imaging of Plant Material ......... 225
David L. Wetzel
7.1 Introduction, Background and Perspective .............. 225
7.1.1 Early Technology ............................... 225
7.1.2 Optical Advances ............................... 226
7.1.3 Early Imaging Applications ..................... 227
7.1.4 Spatial Resolution ............................. 228
7.2 Application of IMS to Kernels and Seeds of Plants ..... 230
7.2.1 Motive and First Applications of Spatially
Resolved IMS ................................... 230
7.2.2 Mapping Tissue Sections ........................ 231
7.2.3 Transitions across Botanical Parts ............. 232
7.2.4 Imaging Single Cells ........................... 234
7.2.5 Applying Synchrotron IMS to Kernels, Seeds
and Other Tissues .............................. 237
7.2.6 Various Applications of IMS .................... 240
7.2.7 Wheat Quality via IMS and Germination Study .... 241
7.2.8 Desiccation Study .............................. 243
7.3 Leaves ................................................ 244
7.4 Application of IMS to Stems and Cellulose Polymers .... 247
7.4.1 Structure and Lignification .................... 247
7.4.2 Application of Polarized IMS ................... 248
7.4.3 Alteration of Cell Wall Architecture ........... 248
7.4.4 Flax Fibers .................................... 250
7.4.5 Biopolymer Structure ........................... 251
7.4.6 Cell Wall Alterations via Mutations ............ 252
7.4.7 Cell Wall Heterogeneity ........................ 253
7.4.8 Esters in Cell Walls ........................... 253
7.5 Algae ................................................. 254
7.6 Comments ......................................... 254
Acknowledgments ............................................ 254
References ................................................. 255
8 Near-Infrared Hyperspectral Imaging in Food and
Agricultural Science ....................................... 259
Véronique Bellon-Maurel cmdjanie Dubois
8.1 Introduction .......................................... 259
8.1.1 A Brief History of Near-Infrared Spectral
Imagers ........................................ 259
8.1.2 When Should NIR Hyperspectral Imaging be Used
in Food and Agricultural Products? ............. 260
8.2 At the Laboratory Scale ............................... 261
8.2.1 Factors to Consider in the Development of
NIR Chemical Imaging Methods ................... 262
8.2.2 The Many Interests of Corn from a NIR Imaging
Perspective .................................... 263
8.2.3 Can NIR Chemical Imaging Replace Traditional
Wet Chemistry? ................................. 266
8.2.4 Interest of NIR Chemical Imaging in Plant
Breeding and GMOs .............................. 267
8.2.5 Interest of NIR Chemical Imaging in Animal
Feed ........................................... 269
8.2.6 A Quick Look at Other Products ................. 270
8.2.7 Conclusion: Laboratory-Based NIR Imaging ....... 271
8.3 At the Industrial Scale ............................... 271
8.3.1 Introduction ................................... 271
8.3.1.1 The Failure of RGB Systems in Food
Quality Control ....................... 271
8.3.1.2 How Did We Come to On-Line NIR
Imaging? .............................. 272
8.3.1.3 When Is NIR Imaging Worth Using in
On-Line Settings? ..................... 274
8.3.2 External Contamination Detection by NIR
Imaging ............................... 275
8.3.2.1 Foreign Bodies ........................ 275
8.3.2.2 Surface Liquid Contaminations ......... 277
8.3.3 Surface and Subsurface Nonconformities ......... 279
8.3.3.1 Human-Detectable Defects .............. 280
8.3.3.2 Potential Defects: Chilling
Injuries, Potential Greening Area ..... 281
8.3.4 Detection of Internal Defects by Candling ...... 282
8.3.4.1 Internal Foreign Bodies ............... 282
8.3.4.2 Internal Tissue Defects ............... 283
8.3.5 Measuring Internal Quality Traits .............. 284
8.3.6 Chemical Imaging Analysis of Heterogeneous
Products and Heterogeneous Product Streams ..... 285
8.3.6.1 Gradual Chemical Changes: Mapping
the Chemistry of a Sample ............. 285
8.3.6.2 Targeted Chemical Composition
Analysis .............................. 287
8.3.7 Conclusion: On-Line Applications ............... 288
8.4 General Conclusions ................................... 289
8.4.1 Hardware Development ........................... 289
8.4.2 Data Processing ................................ 290
8.4.3 Calibration and Characterization of NIR
Imaging Systems ................................ 290
8.4.4 Concluding Remarks ............................. 292
References ................................................. 292
Part Four Polymers and Pharmaceuticals
9 FT-IR and NIR Spectroscopic Imaging: Principles,
Practical Aspects and Applications in Material and
Pharmaceutical Sciences .................................... 297
Elke Wessel, Christian Vogel, Olga Kolomiets, Uwe
Hoffmann, and Heinz W. Siesler
9.1 Introduction .......................................... 297
9.2 Instrumentation for FT-IR and NIR Imaging ............. 299
9.2.1 NIR Imaging .................................... 300
9.2.2 FT-IR Imaging .................................. 302
9.2.2.1 Micro FT-IR Imaging ................... 303
9.2.2.2 Macro FT-IR Imaging ................... 304
9.2.2.3 Measurement of an FT-IR Image ......... 305
9.2.2.4 Observation of a Penetration Depth
Gradient in ATR FT-IR Spectroscopic
Imaging Applications .................. 306
9.2.2.5 Influence of Pressure on ATR FT-IR
Imaging Results ....................... 310
9.2.2.6 Spatial Resolution of Micro FT-IR
Imaging Measurements .................. 312
9.3 Applications of FT-IR Imaging for Polymer Research
and Quality Control ................................... 318
9.3.1 Investigation of Phase Separation in
Biopolymer Blends .............................. 318
9.3.2 Imaging Anisotropic Materials with Polarized
Radiation ...................................... 321
9.3.2.1 Blends of PHB and PLA ................. 321
9.3.2.2 Stress-Induced Phase Transformation
in Poly(vinylidene fluoride) .......... 324
9.3.3 Diffusion of D20 into Polyamide 11 ............. 327
9.3.3.1 Experimental .......................... 328
9.3.3.2 Results and Discussion ................ 329
9.3.4 Conclusions .................................... 334
9.4 NIR Imaging Spectroscopy for Quality Control of
Pharmaceutical Drug Formulations ...................... 335
9.4.1 Quantitative Determination of Active
Ingredients in a Pharmaceutical Drug
Formulation .................................... 335
9.4.2 Spatial Distribution of the Active
Ingredients in a Pharmaceutical Drug
Formulation .................................... 340
9.4.3 Conclusions .................................... 343
References ................................................. 343
10 ATR-FT-IR Imaging for Pharmaceutical and Polymeric
Materials: From Micro to Macro Approaches .................. 347
Sergei C. Kazarian, K.L. Andrew Chan, and Feng H. Tay
10.1 Introduction .......................................... 347
10.2 The Versatility of ATR-FT-IR Imaging .................. 349
10.2.1 Micro ATR Imaging .............................. 351
10.2.2 ATR-FT-IR Imaging with a Diamond Accessory ..... 352
10.2.2.1 Comparison of'New' and 'Old' Diamond
ATR Accessories ....................... 353
10.2.3 ATR-FT-IR Imaging with an Expanded Field of
View ........................................... 354
10.2.4 ATR-FT-IR Imaging with Variable Angle of
Incidence ...................................... 355
10.2.5 Quantitative ATR-FT-IR Imaging ................. 356
10.3 Applications of ATR-FT-IR Imaging in the Materials
Sciences .............................................. 358
10.3.1 Study of the Polymer/Carbon Fiber Interface .... 358
10.3.2 Polystyrene, Polyethylene Blend: The Effect
of a Compatibilizer ............................ 361
10.4 Applications of ATR-FT-IR Imaging in the
Pharmaceutical Sciences ............................... 361
10.4.1 Imaging of Compacted Tablets ................... 361
10.4.2 ATR-FT-IR Imaging of Tablet Dissolution ........ 364
10.4.3 High-Throughput Studies with ATR-FT-IR
Imaging ........................................ 367
10.5 ATR-FT-IR Imaging for Forensic Applications ........... 370
10.5.1 Detection of Trace Materials ................... 370
10.5.2 Imaging of Counterfeit Tablets ................. 372
10.6 Conclusions and Outlook ............................... 372
References ............................................ 373
11 Raman Spectral Imaging on Pharmaceutical Products .......... 377
Eunah Lee
11.1 Overview .............................................. 377
11.2 Hyperspectral Imaging ................................. 378
11.2.1 Terminologies .................................. 378
11.2.2 Advantages ..................................... 378
11.2.3 Spectra versus Image ........................... 379
11.2.3.1 Spectral Intensity and Speed .......... 379
11.2.3.2 SNR and Image Quality ................. 380
11.2.3.3 Spectral Resolution and Detection
Limit ................................. 382
11.2.3.4 Instrumentation ....................... 383
11.2.4 An Example of Raman Hyperspectral Imaging
Analysis: I .................................... 383
11.3 Empirical Approach to Successful Raman Hyperspectral
Imaging ............................................... 383
11.3.1 Spectroscopy ................................... 383
11.3.2 Mapping ........................................ 386
11.3.3 Data Analysis: Spectroscopy .................... 388
11.3.3.1 Unsupervised Analysis ................. 388
11.3.3.2 Supervised Analysis ................... 392
11.3.4 Data Analysis: Raman Images .................... 393
11.3.4.1 Statistical Analysis .................. 393
11.3.4.2 Morphological Analysis ................ 394
11.3.5 An Example of Raman Hyperspectral Imaging
Analysis: II ................................... 396
11.4 Raman in Multimodal Hyperspectral Imaging ............. 397
11.5 Conclusions ........................................... 400
Acknowledgments ............................................ 401
References ................................................. 402
Part Five Developments in Methodology
12 Deep, Noninvasive Raman Spectroscopy of Diffusely
Scattering Media ........................................... 405
Pavel Matousek
12.1 Introduction .......................................... 405
12.2 Techniques for Deep, Noninvasive Raman Spectroscopy ... 406
12.2.1 Temporal Methods: Ultrafast Gating ............. 406
12.2.2 Spatial Methods: Spatially Offset Raman
Spectroscopy (SORS) ............................ 407
12.2.3 Inverse SORS ................................... 410
12.2.4 Transmission Raman Spectroscopy ................ 411
12.2.5 Raman Signal Enhancement Using a Bandpass
Filter ......................................... 412
12.3 Examples of Application Areas ......................... 414
12.3.1 Probing of Bones through Skin for Disease
Diagnosis ...................................... 414
12.3.2 Chemical Identification of Calcifications in
Breast Cancer Lesions .......................... 416
12.3.3 Probing of Pharmaceutical Tablets in Quality
Control ........................................ 419
12.3.4 Probing of Pharmaceutical Capsules in Quality
Control ........................................ 421
12.3.5 Noninvasive Detection of Counterfeit Drugs ..... 421
12.4 Outlook on Raman Tomography .......................... 424
12.5 Conclusions .......................................... 424
Acknowledgments ....................................... 424
References ................................................. 425
13 Planar Array Infrared Spectroscopy and 1-D Imaging ......... 427
Christian Peller'm
13.1 Introduction .......................................... 427
13.2 Concept and Instrumentation ........................... 427
13.3 PA-IR Spectroscopy .................................... 430
13.4 Applications .......................................... 434
13.4.1 Ultrathin Films ................................ 434
13.4.2 Time-Resolved Studies .......................... 437
13.4.3 Dynamic IR Linear Dichroism .................... 442
13.4.4 Emission PA-IR Imaging ......................... 444
13.4.5 PA-IR Microscopy ............................... 447
13.5 Conclusions ........................................... 448
References ............................................ 448
14 Biomedical Applications of Infrared Microspectroscopy
Using Synchrotron Radiation ................................ 451
Lisa M. Miller
14.1 Introduction .......................................... 451
14.2 Spatial Resolution Considerations ..................... 452
14.3 Advantages of a Synchrotron IR Source ................. 454
14.4 Instrumentation ....................................... 455
14.4.1 Infrared Beamlines ............................. 455
14.4.2 Synchrotron Infrared Microscopes ............... 457
14.4.3 Infrared Detectors ............................. 458
14.5 Biological Sample Preparation and Modes of Data
Collection ............................................ 458
14.5.1 Transmission Mode .............................. 459
14.5.2 Reflection Mode ................................ 459
14.6 Biological and Medical Applications of Synchrotron
IR Microspectroscopy .................................. 462
14.7 Future Directions for Synchrotron IR
Microspectroscopy ..................................... 466
Acknowledgments ............................................ 468
Abbreviations .............................................. 468
References ................................................. 468
15 Spectroscopic Imaging with Nanometer Resolution Using
Near-Field Methods ......................................... 473
Boon-Siang Yeo, Thomas Schmid, Weihua Zhang, and
Renato Zenobi
15.1 Introduction .......................................... 473
15.2 Methods ............................................... 474
15.2.1 Scanning Near-Field Optical Microscopy with
Aperture Probes ................................ 474
15.2.2 Tip-Enhanced Raman Spectroscopy ................ 475
15.2.3 Scattering SNOM ................................ 478
15.2.4 Comparison of the Near-Field Spectroscopic
Methods ........................................ 479
15.2.5 Imaging ........................................ 479
15.3 Applications .......................................... 480
15.3.1 Carbon Nanotubes ............................... 480
15.3.2 Semiconductors ................................. 481
15.3.3 Polymers and Other Materials ................... 484
15.3.4 Biological Applications - I: IR a-SNOM of
Fibroblasts .................................... 486
15.3.5 Biological Applications - II: DNA .............. 488
15.3.6 Biological Applications - III: AFMIR of
Escherichia coli ............................... 489
15.3.7 Biological Applications - IV: Towards Full
Spectroscopic Imaging .......................... 491
15.4 Current Challenges .................................... 494
15.4.1 Performance of the Tips ........................ 494
15.4.2 TERS Signal Modulation by Surface Roughness .... 494
15.4.3 Tip Contamination, Analyte Dissociation and
'Blinking' ..................................... 495
15.5 Summary and Outlook ................................... 496
Acknowledgments ....................................... 497
References ............................................ 497
Index ................................................. 501
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