Informatics, digital & computational pathology

Digital and whole slide imaging

WSI fundamentals

Editor-in-Chief: Debra L. Zynger, M.D.
Samreen Fathima, M.B.B.S.
Anil Parwani, M.D., Ph.D., M.B.A.

Last author update: 31 March 2020
Last staff update: 14 May 2021

Copyright: 2019-2024,, Inc.

PubMed Search: Whole slide imaging[TI] free full text[sb]

Samreen Fathima, M.B.B.S.
Anil Parwani, M.D., Ph.D., M.B.A.
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Cite this page: Fathima S, Parwani A. WSI fundamentals. website. Accessed February 22nd, 2024.
Definition / general
  • Whole slide imaging (WSI) or virtual microscopy involves the scanning (digitization) of glass slides to produce digital slides (Adv Anat Pathol 2012;19:152)
Essential features
  • Whole slide imaging has 2 major components: hardware and software
    • Hardware:
      • Microscope with lens objectives
      • Light source (bright field or fluorescent)
      • Robotics to load and move glass slides around
      • Digital cameras for image capture
      • Computer
    • Software to manipulate, manage and view digital slides
  • Static digital image, virtual microscopy

Contributed by Anil Parwani, M.D., Ph.D., M.B.A.

Omnyx whole slide imaging scanner

Tile based and line based scanning

  • First scanners introduced in by Wetzel and Gilbertson in 1999 (Arch Pathol Lab Med 2019;143:222) ¬†
  • Evolved from static digital images and robotic microscopy
  • In 1997, Ferreira et al. used robot / microscope / computer combination to create a mosaic pattern of image tiles that produced a composite slide image (Path Lab Med Int 2015;7:23)
  • A system capable of capturing entire slides at high resolution but in a time efficient fashion and with reasonable operating costs was produced by Interscope Technologies (Path Lab Med Int 2015;7:23)
  • Traditional glass histology slide is digitized via a slide scanner and viewed on a computer screen or handheld device at a similar resolution as light microscopy
  • Robotics are key to avoid breaking slides, for stage accuracy and for dependable objective switching
  • Whole slide imaging instruments do batch scanning (i.e. scan one slide at a time) and continuous or random access processing (i.e. slides can be uploaded while another is being scanned)
  • Entire glass slide or a preselected region of interest on the slide can be scanned
  • Digital data are captured via the camera's charge coupled device (CCD) and then a computer employs specialized imaging software to generate a virtual slide
Common methods of scanning
  • Tile based scanning
    • Robotics controlled motorized slide stage obtains large numbers of square image frames and assembles them into a mosaic pattern
    • Charge coupled device captured tiles are autocorrelated with each other to ensure proper alignment and stitched together in a single, massive seamless image
  • Line based scanning
    • Servomotor based slide stage moves in a jitter free linear fashion on a single axis of acquisition
    • Group of images is produced in the form of long, uninterrupted strips or lines
Techniques used in scanners with slide staining
  • Bright field
    • Emulates standard bright field microscopy and is most common and cost effective
  • Fluorescent
    • Used to digitize fluorescently labeled slides (i.e. fluorescent immunohistochemistry, fluorescent in situ hybridization)
    • Captures images as tiles
  • Multispectral
    • Captures spectral information across the spectrum of light
    • Applied to both the bright field and fluorescent settings
  • Image viewer
    • Software utilized to navigate digital slides
    • Allows users to view and navigate (pan and zoom) virtual slides on a digital screen
  • Image analysis
    • Iterative process of adjusting the algorithm parameters, running the algorithm on a subset of images and¬†evaluating the algorithm performance until sufficient algorithm performance is achieved
Categories of measurements
  • Area based
    • Basic assessments, for example, quantifying the areas (2 dimensional) of a certain stain (e.g. chemical or immunohistochemistry stain), the area of fat vacuoles or other events present on a slide
  • Cell based
    • Identifying and enumerating objects, e.g. cells
    • Enables subsequent assessment of subcellular compartments
Pathologist's role in image analysis workflow
  • To ensure the value and quality of generated data
  • Value: technical knowledge of tissue handling, fixation, processing and staining, as well as the specialty expertise in biology, histology, pathology, pathophysiology, biomarker expression, comparative anatomy, etc.
  • Quality: quality of the tissue, histology slide, stain and scan
  • Validity of image analysis data can be greatly hampered by performing analysis on low quality tissue / histology slides or improperly optimized staining results
  • Preanalytical variables such as interval between tissue harvest and fixation and total fixation time are often poorly controlled
Clinical validation
  • Validation refers to the demonstration of equivalent diagnostic performance between digital slides and glass slides examined
  • According to the College of American Pathologists (CAP), validation of the entire whole slide imaging system that involves trained pathologists should be performed in a manner that emulates the laboratory's actual clinical environment
  • CAP recommends including at least 60 routine cases per application to assess interobserver diagnostic concordance between digitized and glass slides, viewed at least 2 weeks apart (Arch Pathol Lab Med 2013;137:1710)
Clinical applications
  • Primary diagnosis
  • Consultation (second opinions)
  • Remotely interpreting frozen sections
  • Remotely viewing immunostains
  • Showcasing pathology slides at tumor boards
  • Archiving and retrieval
  • Performing image analysis
  • Proficiency training
Nonclinical applications
  • Education
  • Clinical trials / research
  • Customized reporting
  • Digitization
  • Data mining
Recent advances
  • High definition hematoxylin and eosin test for digital pathology
  • Color calibration slide to promote color standardization
  • Vendor neutral viewers (e.g. OpenSlide)
  • Usage of enhancement to avoid tissue folds interference with image analysis algorithms
  • Limiting technology and regulatory issues
  • Image quality
  • Cannot scan all materials (e.g. cytology, microbiology)
  • Cost of the systems
  • Digital slide storage
  • Inability to handle high throughput routine work
  • Regulatory barriers in certain countries
  • User unfriendly ergonomics
  • Increases viewing time
  • Pathologists' reluctance to use whole slide imaging

Whole slide imaging overview

Scanning (MikroScan D2)

Board review style question #1
Tile based scanning is

  1. An iterative process of adjusting the algorithm parameters and running the algorithm on a subset of images
  2. Robotics controlled motorized slide stage that obtains large numbers of square image frames and assembles them into a mosaic pattern
  3. Servomotor based slide stage that moves in a jitter free linear fashion on a single axis of acquisition
  4. Software utilized to navigate digital slides
Board review style answer #1
B. Robotics controlled motorized slide stage that obtains large numbers of square image frames and assembles them into a mosaic pattern

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Reference: Whole slide imaging fundamentals
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