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Spatial multiplexing and omics

Nature Reviews Methods Primers volume 4, Article number: 54 (2024) Cite this article

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An Author Correction to this article was published on 02 September 2024

This article has been updated

Abstract

Much like solving a game of Clue, understanding physiological mysteries involves answering who, what, when and where. Multiomics approaches delve into cellular and molecular identities (the who and what), whereas longitudinal data collection addresses the when. Spatial dimensions address the where. This Primer discusses current technologies enabling quantification across biological scales, emphasizing the importance of retaining the spatial dimension of that data. We outline experimental design considerations, including targeted versus untargeted approaches, sample types, biological scale and four main classes of molecule detection. Spatial analytics are explored, covering questioning approaches, analytical platforms, image segmentation and sampling. Example applications, reproducibility considerations, limitations and our outlook for the future are provided. Our goal is to unite spatial platforms and biological scales that not commonly brought together to encourage collaboration and innovation between diverse biological fields, offering a conceptional framework and an apples-to-apples comparison for understanding major technologies to foster cross-disciplinary dialogue.

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Fig. 1: Biological questioning through a spatial lens.
Fig. 2: Biological scale of common spatial technologies.
Fig. 3: Experimentation tools diagram.
Fig. 4: Acquisition area and marker throughput by commercial platforms and biological scale.
Fig. 5: Technological integration roadmap.

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Acknowledgements

J.L.C. was supported by the University of Alabama at Birmingham start-up funds. A.R. and S.N.K. were supported by CCSG Bioinformatics Shared Resource 5 P30 CA046592, a gift from Agilent technologies and a Precision Health Investigator award from U-M Precision Health to A.R. along with L. Rozek and M. Sartor. S.N.K. and A.R. were partially supported by the NCI Grant R37-CA214955. S.N.K. and A.R. were also partially supported by the University of Michigan (U-M) start-up institutional research funds. S.N.K., S.F.-B. and A.R. were also supported by a Research Scholar Grant from the American Cancer Society (RSG-16-005-01). E.H.S. was supported by a Cancer Prevention and Research Institute of Texas (CPRIT) award RP190617. A.G.S. was supported by NIH funding R01CA279143, R01CA276540, R01CA240589 and the Preclinical Imaging Facility (5P30CA013148).

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Authors and Affiliations

  1. Department of Medicine, Division of Hematology and Oncology, University of Alabama at Birmingham, Birmingham, AL, USA

    Julienne L. Carstens

  2. O’Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA

    Julienne L. Carstens & Anna G. Sorace

  3. Department of Electrical and Computer Engineering, Rice University, Houston, TX, USA

    Santhoshi N. Krishnan & Arvind Rao

  4. Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA

    Santhoshi N. Krishnan & Arvind Rao

  5. Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA

    Arvind Rao

  6. Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA

    Arvind Rao

  7. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Arvind Rao

  8. Department of Radiology, University of Alabama at Birmingham, Birmingham, AL, USA

    Anna G. Sorace

  9. Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL, USA

    Anna G. Sorace

  10. Department of Chemistry, University of Texas at Austin, Austin, TX, USA

    Erin H. Seeley

  11. Department of Gynecologic Oncology and Reproductive Medicine, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Sammy Ferri-Borgogno

  12. Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Jared K. Burks

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  1. Julienne L. Carstens
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  2. Santhoshi N. Krishnan
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Contributions

Introduction (J.L.C., S.F.-B. and J.K.B.); Experimentation (J.L.C., S.N.K., A.R., A.G.S., E.H.S., S.F.-B. and J.K.B.); Results (J.L.C., S.N.K., A.R., S.F.-B. and J.K.B.); Applications (J.L.C. and J.K.B.); Reproducibility and data deposition (J.L.C., A.G.S., E.H.S. and J.K.B.); Limitations and optimizations (J.L.C., A.G.S. and J.K.B.); Outlook (J.L.C., E.H.S., S.F.-B. and J.K.B.); overview of the Primer (J.L.C.).

Corresponding authors

Correspondence to Julienne L. Carstens or Jared K. Burks.

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Competing interests

A.R. serves as a member for Voxel Analytics, LLC and consults for Genophyll, LLC. J.K.B. consults for Standard BioTools, Fortis Labs and Biogenex. All other authors declare no competing interests.

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Nature Reviews Methods Primers thanks Yvonne Vercoulen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Glossary

Antigen retrieval

Process of reversing crosslinking resultant from fixation to make antigens accessible to antibodies. Common methods include heat-mediated retrieval using neutral pH or high pH buffers or enzymatic/proteolytic digestion.

Autofluorescence

The light emitted by biological substances, specifically within 330–500 nm bands, which are often subtracted from optical-based detection methods by default.

Drop-out

A phenomenon in which cells should express a certain marker but do not because the sample was too small to detect it. This is common in single-cell sequencing, but can be applied to any method functioning at the edges of detection.

Image registration

The process in which cyclic or multimodal images have the pixels aligned to match the regions of the same tissue imaged at different times/across serial sections of the same tissue.

Image segmentation

Partitioning of the image into objects or segments (organelle, cell, tissue or organ) that are biologically relevant or significant based on individual image pixel information.

Multiplexing

Analysis of two or more molecules at the same time.

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Carstens, J.L., Krishnan, S.N., Rao, A. et al. Spatial multiplexing and omics. Nat Rev Methods Primers 4, 54 (2024). https://doi.org/10.1038/s43586-024-00330-6

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