For getting the best results and volumetrically correct results out of your histological specimens, please follow these rules and also communicate them to the histotechnician preparing the sections:

  • Choose a meaningful section thickness and number of sections: Cutting thick sections and/or leaving out sections will result in worse reconstruction results, especially for higher magnification levels. E.g., if you are interested in analyzing cells or vascular trees, we recommend to cut sections at a thickness of about 2µm or less (if possible). If the goal is to visualize or measure larger structures like tumors, a section thickness of 20µm might also be sufficient. If in doubt, just cut as thin as possible (without losing too many sections due to ruptures) and try to collect all sections.
  • Cut consecutive sections in equal spacings and thickness. Also write down the thickness and which slices were discarded (if any) in a simple text file that goes with the digitized data. (You might even want to keep corrupt, torn, or otherwise destroyed sections - they can be excluded from the analysis later.)
  • It's OK to place more than one section on a slide. But please place multiple sections on a glass slides in a grid pattern, i.e., align sections horizontally and/or vertically, do not place them diagonally adjacent to each other, and make sure that they don't overlap but instead have some distance between them.
  • Make sure that the section ordering is kept consistent, i.e.
    • sections are placed on the slides in the order they were cut,
    • the ordering scheme within a glass slide is kept consistent (e.g. bottom to top),
    • the glass slides are labeled and placed in a box in the correct order.
    • Try to keep the staining as constant as possible and avoid light stainings.
  • Do not make any markings on the glass slides. The digitization process should be considered part of the specimen preparation and should therefore be performed as the first step right after the glass slides have been produced.
  • Scan all glass slides belonging to one specimen consecutively, in the correct order, and with the same scan settings. Please do not set the scan regions too small, i.e., make sure that the entire sections are scanned, even if you're only interested in a sub-region.
  • Use the scanner vendor's native file format, i.e., avoid .tif (except for Huron/Philips/exported Roche-Ventana), .png, and .jpg output formats. (See also our knowledge base article "Why do I get a warning about a non-optimal file format or unsuitable pyramidal structure?" for more details on this.)
  • Create one folder per subject and include the text file with section thickness / spacing / discarded sections with the data.
  • Do not store several different scans in the same file, i.e. create two separate files for a brightfield and a fluorescence scan of the same slide.