Figure 1.
Confocal Raman spectroscopy of purified hormones.
(A) Raman fingerprint of insulin (a), glucagon (b), amylin (c), somatostatin (d), pancreatic polypeptide (e). Spectra are vertically offset for clear representation. Bands specific for sulfide bridges between cysteines in insulin were found at 520 and 662 cm-1. Tryptophan-specific bands at 759 and 1552 cm-1 were found in spectrum of glucagon. (B) Amino acid composition of insulin (a), containing three Raman-active sulfide bridges between cysteine groups, and glucagon (b), containing the Raman-active amino acid tryptophan.
Figure 2.
Raman spectroscopic evaluation of insulinoma cell lines.
(A) Average Raman fingerprints of MIN6 (a), INS-1E (b) and alpha-TC6 (c) cells were acquired by averaging the spectra of 5 randomly chosen individual cells. Spectra are vertically offset for clear representation. (B) Multivariate data analysis was applied to the dataset to objectively assess subtle differences in the fingerprint region of the spectra. Hierarchical cluster analysis (HCA) classifies the various cell types in separate clusters. (C) PC3 scores from PCA on spectra of the cell cytoplasm clearly distinguish the alpha from the beta cells. Black circles: MIN6 cells, red triangles: INS-1E cells, blue squares: alpha-TC6 cells. (D) PC3 loadings indicate the Raman bands that are mainly involved in the separation of alpha and beta cells. Band positions characteristic for unsaturated fatty acids are marked with an asterisk.
Figure 3.
Label-free Raman imaging of rat insulinoma INS-1E beta-cells.
(A) Brightfield image of a beta-cell. (B) Raman cluster image acquired by scanning the confocal laser beam in a 64x64 raster pattern. Analysis with 3 levels of clustering illustrates the spatial variation of Raman signal within a cell by distinguishing subcellular structures. (C) Corresponding Raman cluster spectra (corrected for background) show the average signal for cytoplasm (magenta) and nucleus (red). Enlargement shows the Raman band at 520 cm-1, specific for difsulfide-bridged cysteine. Spectra are vertically offset for clear representation. Integrating over specific Raman bands shows (D) the distribution of DNA (783 cm-1, Δ = 26 cm-1) corresponding to red cluster in B, and (E) the distribution of disulfide bridges between cysteine groups (524 cm-1, Δ = 30 cm-1). Scale bar represents 10 µm.
Figure 4.
Label-free Raman imaging of primary human islets of Langerhans.
(A) Brightfield microscopy image of a cryosection of an islet of Langerhans. (B) Fluorescence microscopy image of the islet stained for DNA (blue), insulin (red) and glucagon (green). (C) Corresponding Raman cluster image and (D) corresponding Raman cluster spectra, acquired by scanning the confocal laser bundle in a 64x64 raster pattern. Analysis with 3 levels of clustering illustrates the spatial variation of Raman signal within the islet. Enlargement shows the disulfide band at 520 and the tryptophan band at 1552 cm-1. Spectra are vertically offset for clear representation. The bottom panel shows hyperspectral Raman images for (E) DNA (783 cm-1, Δ = 26 cm-1), (F) disulfide bridges between cysteine groups (524 cm-1, Δ = 30 cm-1) and (G) tryptophan (1545 cm-1, Δ = 21 cm-1). Minor artifacts caused by the image-stitching procedure are visible in the Raman images. Scale bar represents 50 µm.
Figure 5.
Label-free detection of insulin and glucagon in human islets.
The top panel shows the univariate Raman images of a cryosection of an islet of Langerhans for (A) DNA (783 cm-1, Δ = 26 cm-1), (B) disulfide bridges between cysteine groups (524 cm-1, Δ = 30 cm-1) and (C) tryptophan (1545 cm-1, Δ = 21 cm-1). The second panel shows the corresponding fluorescence microscopy images stained for (E) DNA, (F) insulin and (G) glucagon. The bottom panel shows the overlay images of the Raman image (in purple) and fluorescence image (green) for (I) DNA, (J) insulin and (K) glucagon. (D) Merged Raman spectroscopy image images of DNA (blue), difsulfide bridged cysteine (red) and tryptophan (green). (H) Merged fluorescence image of DAPI (blue), insulin (red) and glucagon (green). Scale bar represents 50 µm.