Overview of Tissue Imaging Methods

  • Sanjay S. Patel
  • Scott J. RodigEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2055)


The rapidly evolving fields of precision medicine and immuno-oncology are together driving an increasing need for detailed investigation of the tumor immune microenvironment (TIME) in a variety of solid tumors and hematologic neoplasms. The development of targeted therapies that may be efficacious in reprogramming the host immune response to recognize and eliminate tumor cells requires accurate identification of the various inflammatory cells and the spatial relationships between them within the TIME. While currently established techniques enable diagnostic pathologists to routinely interrogate for up to two protein markers and evaluate their expression by visual examination, there is a growing need to reliably query many more targets (i.e., multiplexing) simultaneously in a given tissue specimen, in order to more precisely characterize and distinguish the TIMEs between different tumor types, and between patients. Several technologies aimed at achieving these goals, including multiplex colorimetric immunohistochemistry (mCIHC), multiplex immunofluorescence (mIF), cyclic immunofluorescence (CycIF), multiplexed ion beam imaging (MIBI), codetection by indexing (CODEX), and digital spatial profiling (DSP), are discussed.

Key words

Immuno-oncology Immune microenvironment Multiplex imaging Immunofluorescence Immunohistochemistry 


  1. 1.
    Tumeh PC, Harview CL, Yearley JH et al (2014) PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515(7528):568–571. Scholar
  2. 2.
    Herbst RS, Soria J-C, Kowanetz M et al (2014) Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515(7528):563–567. Scholar
  3. 3.
    Garon EB, Rizvi NA, Hui R et al (2015) Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372(21):2018–2028. Scholar
  4. 4.
    Ansell SM, Lesokhin AM, Borrello I et al (2015) PD-1 blockade with nivolumab in relapsed or refractory Hodgkin’s lymphoma. N Engl J Med 372(4):311–319. Scholar
  5. 5.
    Topalian SL, Hodi FS, Brahmer JR et al (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366(26):2443–2454. Scholar
  6. 6.
    Powles T, Eder JP, Fine GD et al (2014) MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515(7528):558–562. Scholar
  7. 7.
    Nakane PK, Pierce GB (1966) Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histochem Cytochem 14(12):929–931. Scholar
  8. 8.
    Nakane PK (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem 16(9):557–560. Scholar
  9. 9.
    Levenson RM, Mansfield JR (2006) Multispectral imaging in biology and medicine: slices of life. Cytometry A 69(8):748–758. Scholar
  10. 10.
    Remark R, Merghoub T, Grabe N et al (2016) In-depth tissue profiling using multiplexed immunohistochemical consecutive staining on single slide. Sci Immunol 1(1):aaf6925. Scholar
  11. 11.
    Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 70(1):46–58. Scholar
  12. 12.
    Carey CD, Gusenleitner D, Lipschitz M et al (2017) Topological analysis reveals a PD-L1-associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma. Blood 130(22):2420–2430. Scholar
  13. 13.
    Lin J-R, Fallahi-Sichani M, Sorger PK (2015) Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method. Nat Commun 6:8390. Scholar
  14. 14.
    Lin J-R, Fallahi-Sichani M, Chen J-Y, Sorger PK (2016) Cyclic immunofluorescence (CycIF), a highly multiplexed method for single-cell imaging. Curr Protoc Chem Biol 8(4):251–264. Scholar
  15. 15.
    Lin J-R, Izar B, Wang S et al (2018) Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. Elife 7.
  16. 16.
    Bandura DR, Baranov VI, Ornatsky OI et al (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81(16):6813–6822. Scholar
  17. 17.
    Lou X, Zhang G, Herrera I et al (2007) Polymer-based elemental tags for sensitive bioassays. Angew Chem Int Ed Engl 46(32):6111–6114. Scholar
  18. 18.
    Angelo M, Bendall SC, Finck R et al (2014) Multiplexed ion beam imaging of human breast tumors. Nat Med 20(4):436–442. Scholar
  19. 19.
    Lechene C, Hillion F, McMahon G et al (2006) High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 5(6):20. Scholar
  20. 20.
    Giesen C, Wang HAO, Schapiro D et al (2014) Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11(4):417–422. Scholar
  21. 21.
    Keren L, Bosse M, Marquez D et al (2018) A structured tumor-immune microenvironment in triple negative breast Cancer revealed by multiplexed ion beam imaging. Cell 174(6):1373–1387.e19. Scholar
  22. 22.
    Goltsev Y, Samusik N, Kennedy-Darling J et al (2018) Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174(4):968–981.e15. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Pathology, Brigham and Women’s HospitalHarvard Medical SchoolBostonUSA

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