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Caption : pca-generated regions of interest (roi) selection of imaging tof-sims data allows chemical comparison of multiple breast cancer specimens.
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Caption : figure 1. (a) chart of h&e stained images for pre and post chemotherapy treated tissues of luminal a type and basal-like from two patients (b) an increased magnification optical h&e stained image of a selected analysis region. (c) summed cn- and cno- ion image from the tof-sims analysis region corresponding to (b) showing the 25 200 × 200 µm tiles comprising of one stitched patch. white regions seen in the tissue slices can indicate either tears or (most typically when round) fatty acid droplets. all scale bars represent 1 mm.
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Caption : figure 2. (a) pc2 scores plot generated from whole patch data using the negative ions m/z > 200. pc2 shows an overall variance of 13%. (b) pc2 loadings displaying the chemical species that correspond to the whole patch scores. pre-chemotherapeutic tissues shown as blue colored ○ and cyan ●, post- chemotherapeutic tissues shown as colored magenta □ and red ■. the lines above and below the data points indicate 95% confidence intervals.
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Caption : figure 3. (a) an example of an analysis patch at higher magnification with pathologist selected areas on the h&e (blue) (b) pathologist selected areas (black lines) projected onto sum ion image of cn- and cno- (m/z 26.0 and 42.0) (c) representative ion image of tiles removed, shown in black, prior to pca. all scale bars are 200 µm.
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Caption : figure 4. optical and tof-sims camera and ion images showing the heterogeneity between serially cut tissue sections for the entire section and at a specific 1 mm × 1 mm analysis area, outlined in black. (a) initial section 1, an optical h&e stained section before the tof-sims section. (d) h&e optical image at an increased magnification of a selected analysis region. black arrow shows circular structure that becomes absent after this section. (b) section 2, used for tof-sims analysis. tof-sims microscope stitch. (e) summed ion image of cn- and cno- does not display similar structure as observed in (d). (c) section 3, h&e made directly after the tof-sims section. (f) increased magnification optical h&e stained section of selected region displaying a more analogous structure to (e). scale bar in d-f represents 200 µm.
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Caption : figure 5. negative ion image pca results with a variance of 0.66% from an example patch. (a) positive scores image showing the isolation of cellular regions. (b) positive loadings that display the chemical species identified within (a) the cellular scores image. (c) negative scores image or the stromal region. (d) negative loadings showing the chemical species identified within the stromal region. the cellular and stromal regions can be distinguished from the h&e stained image presented in figure 3a. image contrast was enhanced for clearer presentation for publication. scale bars = 200 µm.
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Caption : figure 6. image pca mask workflow. using the scores images that isolate the cellular (a) and stromal (b) areas and subtracting fatty acid (fa) droplets (c), if present within the sample, region specific threshold masks (d and e) can be generated. images can then be reconstructed to contain only spectra representative of the (d) cellular or (e) stromal regions. all scale bars are 200 µm.
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Caption : figure 7. spectral pca results of cellular/tumor areas between tissue samples using image pca masks to reconstruct only cellular/tumor regions for each tissue. (a) pc2 scores generated using pca masks using the negative ions m/z > 200. (b) loadings plot displaying the chemical species that correspond to pca mask analysis scores. this method provides the best separation between the samples when compared to the previous methods, capturing 13% of the total variance. pre-chemotherapy tissues shown as blue colored ○ and cyan ●, post-chemotherapy tissues shown as colored magenta □ and red ■.
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Caption : figure 8. (a) pc1 vs pc2 scores using image pca masks to reconstruct the cellular/tumor regions using negative ions m/z > 200. (b) pc1 loadings plot displaying the chemical species that correspond to pc1 scores (x-axis) (c) pc2 loadings plot displaying the chemical species that correspond to pc2 scores (y-axis). pre-chemotherapeutic tissues shown as blue colored ○ and cyan ●, post-chemotherapeutic tissues shown as colored magenta □ and red ■.