Label-Free Live-Cell Imaging with Confocal Raman Microscopy Katharina Klein, Alexander M. Gigler, Thomas Aschenbrenner, Roberto Monetti, Wolfram Bunk, Ferdinand Jamitzky, Gregor Morfill, Robert W. Stark, Jürgen Schlegel  Biophysical Journal  Volume 102, Issue 2, Pages 360-368 (January 2012) DOI: 10.1016/j.bpj.2011.12.027 Copyright © 2012 Biophysical Society Terms and Conditions

Figure 1 Structural filtering of the PCA components of a Raman spectroscopic image. The upper row shows the components in the usual rank order according to their explained variances. The lower panels are reordered with respect to the anisotropic structural information content. Most prominently, components 1–5 remained in their original order, while components 6–8 were rejected due to the high proportion of artificial structural elements (i.e., systematic errors). Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 2 Matching sequence of a Raman image (by integration over Raman intensities, 80–3040 cm−1) and the corresponding IF data (RGB image). Panel 0 (top row, left) shows the integrated Raman, and panel 8 (bottom row, right) shows the IF image. Panels 1–7 show stepwise overlays demonstrating the quality of image registration achieved by successively changing the respective image contribution. For the registration procedure, the first three (rearranged) PCA components were considered. Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 3 (a) The five different cellular compartments can be distinguished by sets of Raman intensities at selected wavenumbers determined by an information-based approach (for more details, see text). The average spectrum of each respective compartment is shown together with its characteristic barcode. The broad band observed at ∼1100 cm−1 originates from the glass substrate, and the one at 1600 cm−1 originates from the buffer solution (PBS). (b) The curves show the difference between the average spectra of the organelles displayed in panel a. The corresponding barcodes have also been included for the sake of clarity. (In color online.) Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 4 Comparison of aIF images of living (a) and fixed (c) LN18 cells with the corresponding IF image (b). The green channel represents the Golgi apparatus. The overall shape of the cell (cytoskeleton) and the bulk distribution of the specific organelles (Golgi apparatus) are preserved between Raman scanning before and after fixation. The positions of the nucleus and nucleoli in both aIF images are almost unchanged. However, what is probably a fixation artifact can be observed in b and c, affecting the cell in the center. The Raman recording in panel a covers a smaller area compared with b and c. The frame indicates the common part of all images. Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 5 Comparison of original IF images (a–d) and (Raman-based) aIF images (e–h). Generally, the images show high similarities. However, the red channel corresponding to the cytoskeleton differs in that panel a shows high localization, and panel e reveals that actin is actually present throughout the cells. Thus, the correlation between a and e is only 0.36. Another obvious difference is observed between c and g (blue channel): In the IF image the nucleus is inhomogeneously stained, whereas the aIF image reveals the presence of nucleoli at these sites. Yet the correlation is 0.74. (b and f) The images of the Golgi apparatus (green channel) yield a correlation of 0.71. Thus, the RGB overlays in d and h are in great agreement. Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions

Figure 6 IF and aIF images from Raman data. The leftmost column 1 shows the integrated Raman images. Columns 2–4 display the aIF images IC∗, which are sensitive to the signal of the endoplasmic reticulum, mitochondria, and the Golgi apparatus (green channel of the RGB images, from left to right). Among these reconstructions, pictures A2, B3, and C4 should be compared with the respective RGB overlay of IF images in the rightmost column 5. Rows A–C correspond to the three different targets for the green fluorophores. The red (blue) channel of the constructions and the IF images represents the signal of the cytoskeleton (nucleus). Biophysical Journal 2012 102, 360-368DOI: (10.1016/j.bpj.2011.12.027) Copyright © 2012 Biophysical Society Terms and Conditions