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CODEX multiplexed tissue imaging

Nature Reviews Immunology volume 23, page 613 (2023)Cite this article

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Co-detection by indexing (CODEX) is a recently developed and commercially available (Akoya Biosciences) technique of highly multiplexed tissue imaging — visualizing up to 60 markers at the single-cell level1,2. It belongs to a set of re-iterative fluorescent tissue imaging methods. Yet what sets CODEX apart from other comparable approaches, such as cyclic immunofluorescence, multiplex immunohistochemistry and Cell DIVE, is that in any given CODEX staining all the antibodies can be applied to the tissue sample at the same time. This vastly reduces the time needed for staining and aids in epitope preservation, which is hard to maintain during cycles of fluorophore bleaching or antibody stripping. This simultaneous staining is achieved through the use of antibodies with covalently conjugated DNA oligonucleotides (which are later revealed by fluorescent detector oligonucleotides) as primary antibody barcodes.

The key benefits of CODEX come from the use of standard four-colour fluorescent microscopy for imaging. This makes it feasible to image large microarrays of patient biopsy samples using mid-resolution air objectives and hence is high throughput. CODEX can also be run at higher resolution with immersion objectives — for example, to image the smaller cells of mouse tissues or the subcellular proteome at nanometre scales when combined with super-resolution techniques such as stochastic optical reconstruction microscopy (STORM). The standard version of CODEX produces around 1 terabyte of data per 1 square centimetre of tissue. These data are three-dimensional, which is crucial for RNA quantification (distributed as isolated dots of staining across the many planes of the cellular volume) together with protein markers. However, the volumetric data can be difficult and expensive to store and manage. With the development of neural net-assisted segmentation algorithms, recent versions of CODEX allow single plane images, which are fast to acquire and segment and do not take much space or expense to store.

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Fig. 1: The CODEX procedure.

References

  1. Goltsev, Y. et al. Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174, 968–981 (2018).

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  2. Kennedy-Darling, J. et al. Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. Eur. J. Immunol. 51, 1262–1277 (2021).

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA

    Yury Goltsev

  2. Department of Pathology, Stanford University, Stanford, CA, USA

    Garry Nolan

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  1. Yury Goltsev
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  2. Garry Nolan
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Correspondence to Yury Goltsev or Garry Nolan.

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Competing interests

Y.G. and G.N. are among the founders and shareholders of Parhelia Biosciences and Akoya Biosciences.

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Goltsev, Y., Nolan, G. CODEX multiplexed tissue imaging. Nat Rev Immunol 23, 613 (2023). https://doi.org/10.1038/s41577-023-00936-z

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