Historically, microscopy has been a hands-on method that can limit higher throughput options in imaging analysis. More recently, options for data analysis in microscopy have expanded from both the introduction of automated microscopy slide imagers, and the adaptation of microscopy slide techniques to imaging in microplates or other high throughput formats. An example of one of these adaptations is the tissue microarray (TMA), a technique where hundreds of individual tissue cores as small as 0.6mm in diameter and 2-5μM thick can be arrayed on a single microscopy slide allowing increased efficiencies in a number of common histology procedures. Already a high throughput solution, TMAs have proven useful in many applications such as bio banking and archiving of biopsy and other tissue samples, disease diagnosis, classification and grading, quality control, antibody and staining optimization during assay development, and for meeting criteria such as the ‘Validation and Verification of Immune Reactivity of All Classes of IHC’s with a Panel of Normal Tissues or Cells’ required by the U.S. FDA regulatory document Guidance for Submission of Immunohistochemistry Applications to the FDA; Final Guidance for Industry.
TMAs were chosen as a model for demonstrating montage and stitching image optimization at high throughput due to their unique geometry and mounting technique. Following microtome sectioning, paraffinized TMA slices are placed in water and the microscopy slide is dipped under the sheet and lifted up out of the water bath to capture the array. Although there is a window of time when the TMA can be repositioned on the slide, the result of this technique is that even panels of the same number and size of cores may not be mounted with the same center offset on a microscopy slide. Additionally, some TMAs may or may not have marker cores that can be offset from the remaining cores. Further, although BioTek microscopy slide adapters used here have been designed for standard 25 x 75 x 1mm microscopy slides, some tolerance should be included when calibrating x-y offsets for imaging to compensate for possible slide movement during robotic transfers and/or image carrier positioning.