(A)WSIs of kidney allograft glomerulus (Upper left: H&E, Upper right: Methenamine sliver trichrome, Lower: H&E) were scanned with a 40X objective lens, 0.95 N.A. The higher resolution 40X WSI enables recognition of glomerular basement membrane splitting commonly associated with transplant glomerulopathy (arrows in Methenamine silver trichrome stain). Inset shows GBM splitting at higher magnification (see the scale). Inflammatory cell subtypes can be easy recognized (e.g. eosinophils (black arrows), plasma cells (black arrow heads), and lymphocytes infiltrating into tubules (yellow arrows)). The inset shows inflammatory cells at higher magnification (see the scale). Eosinophil granules and the perinuclear hof of plasma cells are clearly evident.
B) WSIs of Masson’s Trichrome-stained liver allograft biopsy scanned using the same conditions as in (A). Upper Panel: Side-by-side comparison of biopsies obtained at 9 months and 36 months after transplantation, as shown here, is of great value in transplantation pathology and more effective and efficient than locating (and switching) glass slides on a traditional microscope. Middle Panel: The WSI viewer also enables a pixel-based color analysis, which can be trained to detect and segment colors of defined properties (e.g. blue fibrosis on trichrome stains) at any WSI magnification (left); higher magnification enables more accurate recognition of fine collagen bundles. The viewer then displays an area mask for user confirmation (right, yellow area) that is extended to the entire WSI. Lower: The WSI fraction of positive pixel area for fibrosis, or a chosen chromogen (e.g. DAB in brown or AEC in red) can be quantified and compared across samples. Color-based techniques combine the ability for a user to outline specific regions of interest (ROI) and measure the relative area of the positive color to the total tissue area; clear vascular spaces and fat globules, etc. are not included.

(A) “Traditional” setup for routine transplant histopathology consists of separate light and fluorescence microscopes usually in different locations with mounted cameras to capture and store region of interest (ROIs). Image capture is limited to ROIs and data management and image analysis are complex and time consuming. Fluorescence microscopy is usually located in a shared-resource facility; slide viewing is limited by photobleaching; and ROI capture, particularly for multiplex stains, is very time consuming and complex; and inconvenient data formats restrict distribution and intuitive use. (B) “Digital” setup converts routinely or multiplex-stained slides into a WSI replacing both the traditional brightfield (H&E, trichrome, etc.) and fluorescence (multiplex) microscopes [(Mirax MIDI (Carl Zeiss, Jena, Germany) combined with a Plan-Apochromat 40x/.95 NA objective lens (Carl Zeiss), a Marlin F-146C Medical camera (arrow head) (ALLIED Vision Technologies, Newburyport, MA), an AxioCam MRm (Carl Zeiss) monochrome high-sensitivity digital charge-coupled device (CCD) camera (arrow) and specifically selected excitation/ emission Qdot filters (Omega Optical, Brattleboro, VT)]. WSI files have a “pyramid” structure: the base layer represents the highest resolution data; other layers are digitally generated to facilitate rapid zooming and panning with a computer mouse. Once a WSI is generated, it can be shared over networks or the internet for discussion and collaboration. Users can review and annotate images anytime, anywhere, at any magnification and for any length of time without the consequence of photobleaching. Moreover, once the image is projected on the computer screen a toolset of image analysis functions becomes available. The pathologist can team with engineers and computer scientists to analyze images and data.

(A) Regions of interest (ROI) can be selected on the WSI at any magnification and individual tiles are exported to FarSight for analysis. In this example, an kidney allograft biopsy is stained for CD45RO (green), CD62L (red), CD3 (yellow), CD8 (cyan), and DAPI (blue). Each fluorescent layer is: 1) exported into grayscale images and imported into FARSIGHT, which 2) segments the nucleus, and 3) orientates the signals around the nucleus to find and represent the spatial relationship between the nucleus and cytoplasmic or cell surface analytes to provide detailed cell phenotypes. 4) The resultant FARSIGHT outcome provides scatter plots and spreadsheet capabilities for data visualization, review, and analysis. Overlays verify cell classification for positive (magenta) and negative (cyan) signals for each analyte in each cell, identified by its center point (centroid). More precise cell phenotype characterization is achievable by creating 3D scatter plots that allow for localization of high, intermediate, and low antigen expression, similar to traditional flow cytometry (left top; 3D plot graph). (B) The program also allows the user to select ROIs either using a box tool or free hand (pen or polygon) tool, and export whole ROIs into each fluorescence layer. Once completed, the images can be imported into FARSIGHT to be analyzed.