Send to

Choose Destination
Elife. 2018 Jul 11;7. pii: e31657. doi: 10.7554/eLife.31657.

Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes.

Lin JR#1,2, Izar B#1,2,3,4, Wang S1,5, Yapp C1, Mei S1,3, Shah PM3, Santagata S1,2,6,7, Sorger PK1,2.

Author information

Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States.
Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, United States.
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States.
Broad Institute of MIT and Harvard, Cambridge, United States.
Harvard Graduate Program in Biophysics, Harvard University, Cambridge, United States.
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, United States.
Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, United States.
Contributed equally


The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high-dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.


cancer biology; computational biology; human; immunopathology; multiplexed imaging; single-cell method; systems biology

Conflict of interest statement

JL, BI, SW, CY, SM, PS, SS No competing interests declared, PS PKS is a member of the Board of Directors of RareCyte Inc., which manufactures the slide scanner used in this study, and co-founder of Glencoe Software, which contributes to and supports open-source OME/OMERO image informatics software. Other authors have no competing financial interests to disclose.

Supplemental Content

Full text links

Icon for eLife Sciences Publications, Ltd Icon for PubMed Central
Loading ...
Support Center