Additional Supporting Information may be found in the online version of this article. Supporting Video may be found at: .

HEP_26131_sm_SuppFig1.tif5708KSupporting Information Figure 1. Panoramic overview of multiplex-stained CK19(green)/β-catenin(cyan)/CD31(red)/αSMA(yellow)/DAPI(blue) WSI of normal human adult male liver showing 10 portal/periportal and 10 perivenular regions of interest (ROIs; yellow squares) selected for comparison analysis of pixel-based quantification using ImageJ (enables single channel analysis) and FarSight (enables multiple channels and tissue cytometry). B) Overviews at low magnification are possible with fluorescently-stained WSI, but not images captured with a traditional fluorescent microscope, because 4X WSI are recreated from images captured at 40X that collect significantly more photons. C) Illustrates the concept of multiple channels/analytes or layers that comprise a multiplex-stained WSI co-localized over particular biologic structures. A traditional fluorescent microscope requires each layer to be captured manually, whereas robots and software accomplish these repetitive tasks on a slide scanner. The red arrow rises through a small bile duct.
HEP_26131_sm_SuppFig2.tif1506KSupporting Information Figure 2. A) Simple pixel-based analysis (e.g. ImageJ) calculates the area occupied by a specific marker, but does not enable identification of complex cell types or numbers or characteristics (See text). B) Cell-based multiple channel analysis enables determination of positive or negative staining for a particular analyte and more precise cell characterization. Uncharacterized cells (see text) were not included so a more direct comparison could be made to the ImageJ analysis in (A). C) Scatter plots of nuclear circularity vs size for perivenular and periportal hepatocytes, BEC, αSMA positive smooth muscle cell (SMC; e.g. vascular smooth muscle cells, portal myofibroblasts, and perhaps occasional stellate cells) and CD31 positive endothelial cell (EC; e.g. sinusoidal, lymphatic, hepatic arterial and portal and central vein endothelium). The bar graph shows that circularity value is higher (close to complete circle = 100) in hepatocytes compared to other cells. In addition, BECs are more circular than CD31 positive cells (ANOVA, p<0.05). Note also the different sizes and profiles of the individual cell populations.
HEP_26131_sm_SuppFig3.tif1447KSupporting Information Figure 3. Schematic explanation of software-based assessment of a WSI. The steps are as follows: 1) Define the threshold for each analyte; positive areas masked yellow enables visual confirmation of the threshold. 2) Nuclear segmentation using parameters, such as size, signal intensity, sensitivity, and intensity cut off, defines each cell and provide a geometric center. 3) Objects/cells are identified by encapsulation number, dilation, minimum encapsulation expression (pixel area), and minimum site expression (pixel area). 4) Collect results (positivity) and analyze, as needed.
HEP_26131_sm_SuppFig4.tif7824KSupporting Information Figure 4. Serial virtual step sectioning panels from Figure 3. Twenty micrometer sections of a normal 50 year-old male human liver was stained with panel A mutiplex [CK19(green)/β-catenin(cyan)/CD31(red)/αSMA(yellow)/DAPI(blue)]. CK19low (green; * terminal bile duct) cell, or Hering Canal cell, that attaches directly to a mature hepatocyte (cyan; three white arrows at 0μm level). Note that the β-catenin is located primarily near the cell membrane. The junction between the two cells is highlighted by the white arrowheads (▸from BEC side, and ◂◂◂ from Hepatocyte side). Note the weak CK19 staining on the side of the CH cell immediately adjacent to the hepatocyte (▸ at 3 and 4.5 μm level).
HEP_26131_sm_SuppFig5.tif3807KSupporting Information Figure 5. Confirmatory single label immunoperoxidase staining (AEC chromogen as red stain) on frozen normal human liver was conducted to verify the presence of HNF4α in BEC lining portal tract bile ducts (upper left; arrows) and HNF1β in periportal hepatocytes (upper right; arrows). A majority of periportal hepatocytes are positive for HNF4α (upper left; white arrowheads), as expected, but occasional hepatocyte are negative for HNF4α (black arrowheads). Most of bile ducts are positive for HNF1β, as expected, but HNF1β expression levels are obviously different in one to another cell (upper right; black arrowheads). CK19 staining in CH cells (lower left panel) also showed focal labeling. The negative staining control is shown in the lower right panel. HC: hepatocyte, BD: bile duct.
HEP_26131_sm_SuppTabs.rtf93KSupporting Information Tables

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