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6 Widefield Raman Imaging of Cells and Tissues

Shona Stewart, Janice Panza, and Amy Drauch . . . . . . . . 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 Widefi eld Raman
Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
6.5.1 Principal Component Analysis . . . . . 171
6.5.2 Mahalanobis and Euclidean
Distance . . . . . . . . . . . . . . . . . . . . . . . . 173
6.5.3 Spectral Mixture Resolution . . . . . . . 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 Quantifi cation of
Carotenoid Antioxidants in the Human Retina
and Skin Mohsen Sharifzadeh, Igor V. Ermakov,
Paul S. Bernstein, and Werner Gellermann . . . . . . . . . . 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 Spatially Resolved Resonance Raman
Imaging of Macular Pigment—Methodology
and Validation Experiments . . . . . . . . . . . . . . 205