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The emerging landscape of spatial profiling technologies

Nature Reviews Genetics volume 23, pages 741–759 (2022)Cite this article

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Abstract

Improved scale, multiplexing and resolution are establishing spatial nucleic acid and protein profiling methods as a major pillar for cellular atlas building of complex samples, from tissues to full organisms. Emerging methods yield omics measurements at resolutions covering the nano- to microscale, enabling the charting of cellular heterogeneity, complex tissue architectures and dynamic changes during development and disease. We present an overview of the developing landscape of in situ spatial genome, transcriptome and proteome technologies, exemplify their impact on cell biology and translational research, and discuss current challenges for their community-wide adoption. Among many transformative applications, we envision that spatial methods will map entire organs and enable next-generation pathology.

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Fig. 1: Illustrative examples of spatial profiling measurements.
Fig. 2: Spatial indexing strategies.
Fig. 3: An overview of imaging-based spatial transcriptomics methods and their performance.
Fig. 4: An overview of imaging-based spatial proteomics methods.

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Acknowledgements

The authors thank M. Elosúa-Bayes and F. Björklund for support with the figure design. J.R.M. acknowledges funding from the Pew Charitable Trusts, the Helmsley Charitable Trust and the NIH (R01 GM143277, R21 CA249728). E.L. acknowledges funding from the Knut and Alice Wallenberg Foundation (KAW 2021.0346, KAW 2021.0189, KAW 2018.0172), Erling Persson Foundation, the NIH (U01 DK120447) and EU Horizon 2020 (EPIC-XS grant 823839 and ESPACE grant 874710). H.H. received support for the project PID2020-115439GB-I00 funded by MCIN/AEI/ 10.13039/501100011033. This publication is also supported as part of a project (BCLLATLAS and ESPACE) that has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (grant agreement nos. 810287 and 874710).

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Authors and Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    Jeffrey R. Moffitt

  2. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Jeffrey R. Moffitt

  3. Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden

    Emma Lundberg

  4. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Emma Lundberg

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

    Emma Lundberg

  6. Chan Zuckerberg Biohub, San Francisco, San Francisco, CA, USA

    Emma Lundberg

  7. CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    Holger Heyn

  8. Universitat Pompeu Fabra (UPF), Barcelona, Spain

    Holger Heyn

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  1. Jeffrey R. Moffitt
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  2. Emma Lundberg
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  3. Holger Heyn
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The authors contributed equally to all aspects of the article.

Corresponding author

Correspondence to Holger Heyn.

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Competing interests

J.R.M. is a co-founder of, consultant for and scientific advisory board member of Vizgen, Inc. E.L. is adviser for Pixelgen technologies and Moleculent. H.H. is co-founder of Omniscope and scientific advisory board member of MiRXES.

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Glossary

Multiplexing

The simultaneous profiling of multiple gene transcripts or proteins through the application of pools with panels of detection probes or antibodies, respectively.

Deterministic barcoding

Flexible profiling of spatial coordinates through the targeted labelling of cells or regions of interest using molecular probes, cell tags or fluorescent markers.

Detection efficiency

In the context of image-based transcriptomics, this concept refers to the fraction of targeted molecules that are present in the sample that are actually detected. As single-molecule fluorescence in situ hybridization (smFISH) is often used as a proxy for ground-truth expression, this quantity is often calculated by comparison with smFISH, though comparison with single-cell RNA sequencing is also common. As there is no standard definition in the field, care should be taken in comparing values.

Formalin-fixed paraffin-embedded

(FFPE). Preservation method for tissues in long-term archival storage. After a tissue is collected, it is preserved through formalin fixation to preserve its spatial architecture and finally embedded in a paraffin wax block.

DNA nanoballs

(DNBs). Dense clusters of DNA amplified by rolling circle replication used for DNA sequencing or spatial RNA capture.

Rolling circle amplification

(RCA). A process by which a circularized single-stranded DNA, such as a padlock probe, is replicated into a long molecule composed of concatenated copies of the circularized probe by extending a bound primer with a polymerase capable of strand displacement.

Cluster density

Distance of clusters generated by local clonal oligonucleotide amplification within a sequencing flow cell.

Combinatorial indexing

Multistep split-pool process to index molecules with a unique combination of barcodes for subsequent identification of spatial location (spatial index) or cell identity (single-cell index).

Padlock probes

Single-stranded DNA or locked nucleic acid oligonucleotide probes hybridized to a target of interest such that the 3′ and 5′ ends align on the target molecule, allowing these ends to be ligated to circularize the probe.

Detection efficiency

Capacity to capture or label RNA, DNA or protein molecules to allow subsequent quantification.

SNAIL probes

Probes that contain a primer probe and a padlock probe, both of which hybridize to the same RNA, placing both probes in proximity such that the 3′ and 5′ end of the padlock can hybridize to the primer probe, allowing the circularization of the padlock via DNA-templated ligation.

Expansion microscopy

An effective super-resolution optical microscopy technique that increases effective optical resolution by attaching the signal of interest to a charged hydrogel that is then physically and isotropically expanded.

Combinatorial barcoding

This process involves the use of barcodes in which the value of individual barcode elements, e.g. ‘1’ values in individual bits of binary barcodes, are shared between multiple targets with targets discriminated by the unique combination of barcode elements, e.g. ‘101’ versus ‘110’.

Error-robust and correcting barcoding schemes

Barcoding schemes in which extra barcode elements are added that allow the detection of barcode elements corrupted in the measurement process and, for some errors, the identification of the correct value for the corrupted element.

Oligopools

Collections of tens to hundreds of thousands of unique, custom oligonucleotide sequences generated inexpensively but in small quantities by array-based methods.

Pseudocolours

Collections of images in which each molecule is fluorescent in only one image with that image representing the pseudocolour, i.e. n pseudocolours can be thought of as n-bit barcodes in which only one bit contains a ‘1’.

Hybridization chain reaction

(HCR). An amplification approach in which two meta-stable, fluorescently labelled DNA hairpins are used to polymerize a repeating structure from these hairpins.

Branched DNA amplification

(bDNA). An amplification approach that leverages a DNA oligonucleotide ‘amplifier’ that contains a targeting sequence in combination with multiple copies of a secondary binding site, effectively amplifying fluorescent signals by converting one binding site into multiples. Iterative rounds of amplification are possible.

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Moffitt, J.R., Lundberg, E. & Heyn, H. The emerging landscape of spatial profiling technologies. Nat Rev Genet 23, 741–759 (2022). https://doi.org/10.1038/s41576-022-00515-3

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