A hematoxylin and eosin (H&E) stained glass slide was first digitized at a 0.3 um resolution with a microscope-based slide scanner (NA 1.3, x40 objective). The slide was subsequently destained with a procedure that removes H&E-staining completely.

The same slide was then immuno-stained with a cocktail of p63 (clone 4A4+Y4A3, dilution 1:200, Novocastra Inc, Newcastle, UK) and AMACR (clone P504S, dilute 1:200, Dako Cytomation, Copenhagen, Denmark) antibodies, and the slide was scanned a second time.

Thus 2 virtual slides (VS) were created for each glass slide.

In the browser, the synchronized VSs are placed in separate layers. By using a 50-50% blending of the layers, the alignment can easily be evaluated.


Each compressed VS has a header, which contains information for exact positioning of the VS into a (pixel-based) coordinate system in the viewer on the computer screen.


If the slides initially are not perfectly aligned, the required adjustment can quickly be measured in the browser with the ruler tool, by clicking a landmark (a distinct cell), first in the H&E, and then in the IHC VS.




The required x and y adjustment is displayed and entered into the header. The start coordinates of the H&E VS are fixed (0,0), while the start coordinates of the IHC VS are adjusted so that exact alignment is achieved. In all, only a few seconds are required for the alignment process.

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