1 High-Resolution Optical and Confocal Microscopy .............. 1
Olaf Hollricher and Wolfram Ibach
1.1 Introduction ............................................ 1
1.2 Introduction to Theoretical Considerations in High-
Resolution Microscopy .................................. 3
1.3 Introduction to Confocal Microscopy ..................... 3
1.4 Electromagnetic Scattering in Optical Systems ........... 4
1.5 3D-Intensity Distribution in the Focus .................. 5
1.5.1 Large Aperture Angles ............................ 6
1.5.2 Transition to Small Aperture Angles .............. 9
1.6 Theory of Image Formation .............................. 11
1.6.1 Microscope ...................................... 11
1.6.2 Confocal Microscope ............................. 12
1.6.3 Confocal Raman Microscope ....................... 12
1.7 Image Formation for Light Scattering ................... 13
1.7.1 Scattering Point ................................ 13
1.7.2 Reflection at a Mirror .......................... 15
1.8 Image Formation for Raman Scattering ................... 16
1.8.1 Raman Emitting Point ............................ 16
1.8.2 Raman Emission of a Layer ....................... 17
1.9 Pinhole Size ........................................... 18
References .................................................. 20
2 Introduction to the Fundamentals of Raman Spectroscopy ...... 21
Benjamin Dietzek, Dana Cialla, Michael Schmitt, and Jürgen
Popp
2.1 Introduction ........................................... 21
2.2 Classical Picture of Light Scattering .................. 22
2.2.1 Frequency Dependence of Raman Scattering ........ 22
2.2.2 Classical "Selection Rule" and Comparison to
IR Absorption ................................... 24
2.2.3 Scattered Raman Intensity ....................... 25
2.2.4 Shortfalls of the Classical Picture ............. 27
2.3 Raman Cross-Section Enhancement Mechanisms ............. 27
2.3.1 Resonant Raman Scattering ....................... 28
2.3.2 Advantages and Applications of Resonance
Raman Scattering ................................ 31
2.3.3 Surface-Enhanced Raman Scattering ............... 34
References .................................................. 40
3 Raman Instrumentation for Confocal Raman Microscopy ......... 43
Olaf Hollricher
3.1 Introduction ........................................... 43
3.2 The Development of Raman Microscopes ................... 44
3.3 Confocality ............................................ 45
3.4 Throughput of a Confocal Raman Microscope .............. 46
3.4.1 Laser Wavelength ................................ 47
3.4.2 Excitation Power ................................ 47
3.4.3 Objective ....................................... 48
3.4.4 Microscope Throughput ........................... 48
3.4.5 Coupling Between Microscope and Spectrometer .... 49
3.4.6 Spectrometer Throughput ......................... 49
3.4.7 Gratings ........................................ 50
3.4.8 CCD Detector .................................... 51
3.5 Conclusion ............................................. 60
References .................................................. 60
4 Software Requirements and Data Analysis in Confocal
Raman Microscopy ............................................ 61
Thomas Dieing and Wolfram Ibach
4.1 Introduction ........................................... 61
4.2 Requirements for Data Acquisition Software ............. 62
4.2.1 Data Acquisition ................................ 62
4.2.2 Correlation of Spatial and Spectral Data ........ 63
4.3 Description of the Data Sets Acquired in Confocal
Raman Microscopy ....................................... 65
4.4 Pre-processing of Raman Spectra ........................ 66
4.4.1 Cosmic Ray Removal .............................. 66
4.4.2 Smoothing ....................................... 68
4.4.3 Background Substraction and Subtraction of
Reference Spectra ............................... 70
4.5 Image Generation ....................................... 72
4.5.1 Univariate Image Generation ..................... 72
4.5.2 Multivariate Image Generation ................... 75
4.6 Image Masks and Selective Average Calculation .......... 81
4.7 Combination of Single Spectra with Multi-spectral
Data Sets .............................................. 82
4.7.1 Basis Spectra ................................... 83
4.7.2 Fitting Procedure ............................... 83
4.8 Combination of Various Images .......................... 84
4.9 The Law of Numbers ..................................... 85
4.10 Materials and Methods .................................. 88
References .................................................. 89
5 Nano-spectroscopy of Individual Carbon Nanotubes and
Isolated Graphene Sheets .................................... 91
Alain Jungen
5.1 Introduction ........................................... 91
5.2 Individual Carbon Nanotubes ............................ 92
5.2.1 Phonons ......................................... 92
5.2.2 Theory .......................................... 95
5.2.3 Experiment ...................................... 96
5.2.4 Microscopy ...................................... 98
5.2.5 Thermography ................................... 100
5.3 Isolated Graphene Sheets .............................. 101
5.3.1 Theory ......................................... 101
5.3.2 Experiment ..................................... 102
5.3.3 Charge Distributions ........................... 105
5.4 Conclusion ............................................ 107
References ................................................. 108
6 Raman Spectroscopy and Confocal Raman Imaging in
Mineralogy and Petrography ................................. 111
Marc Fries and Andrew Steele
6.1 Introduction .......................................... 111
6.2 Raman Spectroscopy and Imaging as a Mineralogy/
Petrography Tool ...................................... 112
6.2.1 Working with Thin Sections ..................... 114
6.2.2 Control of Laser Power ......................... 116
6.3 "Raman Mineralogy" Using Imaging Raman Techniques ..... 119
6.3.1 Mineral Phase Imaging .......................... 120
6.3.2 Crystallographic Orientation Imaging ........... 122
6.3.3 Phase Composition Imaging ...................... 123
6.4 Examples of "Raman Petrography" Applications .......... 124
6.4.1 Raman Analysis of Shocked Minerals ............. 124
6.4.2 Contextual Imaging of Carbonaceous Materials ... 126
6.4.3 Fluid Inclusions ............................... 129
6.4.4 Ancient Terrestrial Carbonaceous Materials ..... 130
6.5 Raman Mineralogy in Field Geology Studies ............. 130
6.5.1 Extraterrestrial Exploration ................... 132
6.6 Conclusion ............................................ 133
References ................................................. 133
7 Raman Micro-spectral Imaging of Cells and Intracellular
Drug Delivery Using Nanocarrier Systems ................... 137
Christian Matthäus, Tatyana Chernenko, Luis Quintero,
Miloš Miljković, Lara Milane, Amit Kale, Mansoor Amiji,
Vladimir Torchilin and Max Diem
7.1 Introduction .......................................... 137
7.2 Method ................................................ 140
7.2.1 Data Acquisition ............................... 140
7.2.2 Introduction to Data Processing Methods ........ 140
7.2.3 Experimental ................................... 144
7.3 Results ............................................... 145
7.3.1 Cell Imaging ................................... 145
7.3.2 Drag Delivery Systems .......................... 152
7.4 Discussion ............................................ 161
References ................................................. 162
8 Confocal Raman Microscopy in Pharmaceutical Development .... 165
Thomas F. Haefele and Kurt Paulus
8.1 Introduction .......................................... 165
8.1.1 Vibrational Spectroscopy in Pharmaceutical
Development .................................... 166
8.1.2 Imaging in Pharmaceutical Development .......... 169
8.1.3 Chemical Imaging in Pharmaceutical
Development .................................... 172
8.1.4 Confocal Raman Microscopy in Pharmaceutical
Development .................................... 176
8.2 Applications of Confocal Raman Microscopy in
Pharmaceutical Development ............................ 179
8.2.1 Practical Considerations ....................... 179
8.2.2 Investigation of Solid Dosage Forms by
Chemical Imaging ............................... 185
8.3 Conclusions ........................................... 198
8.4 Materials and Methods ................................. 198
References ................................................. 199
9 Characterization of Therapeutic Coatings on Medical
Devices .................................................... 203
Klaus Wormuth
9.1 Background ............................................ 203
9.2 Passive Therapeutic Coatings .......................... 205
9.2.1 Coating Thickness .............................. 205
9.2.2 Swelling of Hydrophilic Gel Coatings ........... 208
9.3 Active Therapeutic Coatings ........................... 210
9.3.1 Coating Morphology ............................. 210
9.3.2 Drug Mixed with Single Polymer: Morphology
and Elution .................................... 211
9.3.3 Drug Mixed with Two Polymers: Blending
and Layering ................................... 216
9.3.4 Drag Mixed with Two Polymers: Exposure to
Water .......................................... 219
9.3.5 Drag Mixed with Biodegradable Polymer: Drug
Elution with Polymer Degradation ............... 220
9.4 Summary ............................................... 223
References ................................................. 223
10 Raman Imaging of Plant Cell Walls .......................... 225
Notburga Gierlinger
10.1 Introduction .......................................... 225
10.2 Plant Cell Walls ...................................... 226
10.3 Micro-Raman Spectroscopy of Plant Fibers .............. 227
10.4 Plant Cell Wall Imaging by Confocal Raman Microscopy .. 228
10.4.1 Imaging Cellulose Orientation and Lignin
Distribution in Wooden Cell Walls .............. 228
10.4.2 Silica and Cell Wall Composition in Horsetail
(E. hyemale) ................................... 232
10.5 Outlook ............................................... 234
10.6 Material and Methods .................................. 234
References ................................................. 235
11 Confocal Raman Imaging of Polymeric Materials .............. 237
Ute Schmidt, Jörg Müller, and Joachim Koenen
11.1 Introduction .......................................... 237
11.2 Raman Imaging of Isotactic Polypropylene (iPP) ........ 239
11.3 Raman Imaging of Polymer Blends ....................... 244
11.3.1 Raman Imaging of Thin Films of the Polymer
Blend: Polystyrene (PS)- Ethyl-Hexyl-Acrylate
(EHA) .......................................... 245
11.3.2 Raman Imaging of Thin Films of the Polymer
Blend Ethyl-Hexyl-Acrylate (EHA)-Styrene-
Butadiene Rubber (SBR) ......................... 247
11.3.3 Raman Imaging of Thin Films of the Polymer
Blend PS-EHA-SBR ............................... 249
11.4 Polymer Coatings ...................................... 251
11.4.1 Acrylic Paints ................................. 251
11.4.2 Adhesives ...................................... 253
11.5 Additives in Polymer Matrices ......................... 253
11.6 Summary ............................................... 257
References ................................................. 257
12 Stress Analysis by Means of Raman Microscopy ............... 259
Thomas Wermelinger and Ralph Spolenak
12.1 Introduction .......................................... 259
12.1.1 Theoretical Background ......................... 260
12.1.2 Measuring in Conventional Back-Scattering
Configuration .................................. 261
12.1.3 Off-Axis Raman Spectroscopy .................... 263
12.1.4 Stress Tensor Analysis in Back-Scattering
Raman Microscopy ............................... 266
12.2 Case Studies .......................................... 268
12.2.1 3D Raman Spectroscopy Measurements ............. 268
12.2.2 ZnO ............................................ 271
12.2.3 The Influence of Stress on the Peak Position
of Polymers .................................... 273
12.3 Discussion ............................................ 276
References ................................................. 277
Index ......................................................... 279
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