Vibrational Spectroscopic Imaging for Biomedical Applications

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Vibrational Spectroscopic Imaging for Biomedical Applications

Vibrational Spectroscopic Imaging for Biomedical Applications

Authors: Gokulakrishnan Srinivasan

Published: August 2010

eISBN: 9780071597081 0071597085 | ISBN: 9780071596992

Contents

Contributors

Preface

1 Toward Automated Breast Histopathology Using Mid-IR Spectroscopic Imaging

1.1 Introduction

1.1.1 FT-IR Imaging

1.1.2 FT-IR Spectroscopic Characterization of Cells and Tissues

1.1.3 FT-IR Imaging for Pathology

1.1.4 High-Throughput Sampling

1.1.5 Modified Bayesian Classification and Automated Tissue Histopathology

1.2 Materials and Methods

1.2.1 Models for Spectral Recognition and Analysis of Class Data

1.2.2 Automated Metric Selection and Classification Protocol Optimization

1.2.3 Spectral Metrics and Biochemical Basis

1.2.4 Validation and Dependence on Experimental Parameters

1.2.5 Application for Cancer Pixel Segmentation

1.2.6 Application for Patient Cancer Segmentation

1.3 Conclusions

References

2 Synchrotron-Based FTIR Spectromicroscopy and Imaging of Single Algal Cells and Cartilage

2.1 Introduction

2.2 IR Environmental Imaging

2.2.1 Beamline Design and Implementation

2.2.2 Initial Measurements with IRENI

2.3 Flow Cell for In Vivo IR Microspectroscopy of Biological Samples

2.3.1 Flow Chamber Design

2.3.2 Mid-IR and Vis Measurements

2.3.3 Viability Tests: PAM Fluorescence Measurements

2.3.4 Initial Flow Cell Measurements with IRENI

2.4 Biomedical Application: Calcium-Containing Crystals in Arthritic Cartilage

2.4.1 Calcium-Containing Crystals and Arthritis

2.4.2 Current Methods of Crystal Identification

2.4.3 Biologic Models of Calcium- Containing Crystal Formation

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

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

Acknowledgments

References

3 Preparation of Tissues and Cells for Infrared and Raman Spectroscopy and Imaging

3.1 Introduction

3.2 Tissue Preparation

3.2.1 Archived Tissue: Paraffin Embedded and Frozen Specimens

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

3.2.3 The Effects of Xylene on Fixed Tissue and Deparaffinization of Paraffin-Embedded Tissue

3.3 Cell Preparation

3.3.1 Chemical Fixation for FTIR and Raman Imaging

3.3.2 Sample Preparation for Biomechanistic Studies

3.3.3 Growth Medium and Substrate Effects on Spectroscopic Examination of Cells

3.3.4 Preparation of Living Cells for FTIR and Raman Studies

3.4 Summary

Acknowledgments

References

4 Evanescent Wave Imaging

4.1 Introduction

4.2 Theoretical Considerations

4.3 Historical Development

4.4 Experimental Implementation

4.5 Benefits of ATR Microspectroscopic Imaging for Biological Sections

4.6 Macro ATR Imaging

4.7 ATR Microspectroscopic Raman Imaging

4.8 Conclusions

References

5 sFTIR, Raman, and SERS Imaging of Fungal Cells

5.1 Introduction

5.2 Introduction to Fungi

5.2.1 Specimen Preparation

5.3 Vibrational Spectroscopy

5.3.1 Spectral Resolution

5.3.2 Spatial Resolution

5.4 sFTIR Spectra of Fungi

5.4.1 Physical Considerations and Spectral Anomalies in sFTIR Spectra

5.5 Raman Spectroscopy of Fungi

5.5.1 Raman Map from a Hypha, at Growing Tip

5.5.2 Raman Map of Spore Branch

5.5.3 Detection of Crystalline Materials by IR and Raman

5.6 SERS Discovery and Development

5.6.1 Substrates: The Key to SERS Imaging

5.6.2 SERS: Applications for Fungi

5.7 Conclusions: Lessons Learned, Caveats, Challenges, Promise

Acknowledgments

References

6 Widefield Raman Imaging of Cells and Tissues

6.1 Introduction

6.2 Generation of Raman Images

6.2.1 Point Mapping

6.2.2 Line Mapping

6.2.3 Other Modes of Generating Raman Images

6.2.4 Widefield Imaging

6.3 Raman Imaging of Cells and Tissues

6.4 Background and Image Preprocessing Steps for Widefield Raman Images

6.4.1 Fluorescence

6.4.2 Correction for Dark Current

6.4.3 Cosmic Filtering

6.4.4 Instrument Response Correction

6.4.5 Flatfielding

6.4.6 Baseline Correction

6.4.7 Normalization

6.4.8 Smoothing

6.5 Chemometric Analysis of Widefield Raman Images

6.5.1 Principal Component Analysis

6.5.2 Mahalanobis and Euclidean Distance

6.5.3 Spectral Mixture Resolution

6.5.4 Derivatives

6.6 Chemometrics in the Analysis of Non-Widefield Raman Images

6.6.1 PCA

6.6.2 Linear Discriminant Analysis

6.7 Conclusions

References

7 Resonance Raman Imaging and Quantification of Carotenoid Antioxidants in the Human Retina and Skin

7.1 Introduction

7.2 Optical Properties and Resonance Raman Scattering of Carotenoids

7.3 Spatially Integrated Resonance Raman Measurements of Macular Pigment

7.4 Spatially Resolved Resonance Raman Imaging of Macular Pigment—Methodology and Validation Experiments

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

7.6 Raman Detection of Carotenoids in Living Human Skin

7.7 Conclusions

References

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

8.1 Introduction

8.2 Raman Imaging of Tissue

8.2.1 Mouse Brains

8.2.2 Human Brain Tumors

8.2.3 Human Colon Tissue

8.2.4 Human Lung Tissue

8.3 Raman Imaging of Cells

8.3.1 Lung Fibroblast Cells

8.3.2 Red Blood Cells

8.4 Raman Spectroscopy of Bacteria

8.4.1 Species Classification

8.4.2 Imaging Single Bacteria

8.5 Conclusions

Acknowledgments

References

9 The Current State of Raman Imaging in Clinical Application

9.1 Introduction

9.1.1 History

9.1.2 Principles

9.2 Instrumentation

9.2.1 Laser

9.2.2 Microscope

9.2.3 Filters

9.2.4 Spectrometer

9.2.5 CCD

9.3 Imaging Techniques

9.4 Data Analysis: Spectra to Image(s)

9.4.1 Classification Techniques

9.4.2 Quantification Techniques

9.5 Raman Mapping and Imaging in Bioscience

9.5.1 Single Cells

9.5.2 Tissues

9.6 Limitations and Perspectives

References

10 Vibrational Spectroscopic Imaging of Microscopic Stress Patterns in Biomedical Materials

10.1 Introduction

10.2 Principles of Raman Spectroscopy

10.3 Raman Effect in Biological and Synthetic Biomaterials

10.3.1 Spectral Features

10.3.2 PS Behavior

10.4 Visualization of Microscopic Stress Patternsin Biomaterials

10.4.1 Micromechanics of Fracture and Crack-Tip Stress Relaxation Mechanisms

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

10.5 Conclusions

References

11 Tissue Imaging with Coherent Anti-Stokes Raman Scattering Microscopy

11.1 From Spontaneous to Coherent Raman Spectroscopy

11.2 The Birth of CARS Microscopy

11.2.1 First Generation CARS Microscopes

11.2.2 Second Generation CARS Microscopes

11.3 CARS Basics

11.3.1 Nonlinear Electron Motions

11.3.2 Resonant and Nonresonant Contributions

11.4 CARS by the Numbers

11.4.1 Signal Generation in Focus with Pulsed Excitation

11.4.2 Photodamaging

11.4.3 CARS Chemical Selectivity

11.4.4 CARS Sensitivity

11.5 CARS and the Multimodal Microscope

11.6 CARS in Tissues

11.6.1 Focusing in Tissues

11.6.2 Backscattering in Tissues

11.6.3 Typical Endogenous Tissue Components

11.7 CARS Biomedical Imaging

11.7.1 Ex Vivo Nonlinear Imaging

11.7.2 In Vivo Nonlinear Imaging

11.8 What Lies at the Horizon?

Acknowledgments

References

Index