Multiplexed spatial transcriptomics methods and the application of expansion microscopy
Open accessAbstract
While spatial transcriptomics has undeniably revolutionized our ability to study cellular organization, it has driven the development of a great number of innovative transcriptomics methods, which can be classified into in situ sequencing (ISS) methods, in situ hybridization (ISH) techniques, and next-generation sequencing (NGS)-based sequencing with region capture. These technologies not only refine our understanding of cellular processes, but also open up new possibilities for breakthroughs in various research domains. One challenge of spatial transcriptomics experiments is the limitation of RNA detection due to optical crowding of RNA in the cells. Expansion microscopy (ExM), characterized by the controlled enlargement of biological specimens, offers a means to achieve super-resolution imaging, overcoming the diffraction limit inherent in conventional microscopy and enabling precise visualization of RNA in spatial transcriptomics methods. In this review, we elaborate on ISS, ISH and NGS-based spatial transcriptomic protocols and on how performance of these techniques can be extended by the combination of these protocols with ExM. Moving beyond the techniques and procedures, we highlight the broader implications of transcriptomics in biology and medicine. These include valuable insight into the spatial organization of gene expression in cells within tissues, aid in the identification and the distinction of cell types and subpopulations and understanding of molecular mechanisms and intercellular changes driving disease development.
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Emerging spatial transcriptomics platforms allow high-resolution, multiplexed mapping of RNA molecules within intact 3D cultures.
“Emerging spatial transcriptomics platforms, such as MERSCOPE (Vizgen), FISSEQ, ExSeq, Slide-seq, and 10x Genomics Visium, now allow for high-resolution, multiplexed mapping of RNA molecules within intact 3D cultures [199,200,201].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [199]
The Introduction says spatial transcriptomics can locate many RNAs through multiplex imaging, and the Methods section describes spatial resolution for Slide-seq. But the paper does not establish that all the listed platforms map RNA within intact 3D cultures: its Methods section describes Slide-seq using sliced tissue, while Table 3 lists organoids for FISSEQ but tissues for Visium. (LLM, conf=0.87)
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Du, J., Yuan, X. H., Deng, H., Huang, R., Liu, B., Xiong, T., Long, X., Zhang, L., Li, Y., & She, Q. (2023) · Journal of Pharmaceutical Analysis · doi:10.1016/j.jpha.2023.07.011 · Cited by 36 · see citing works
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Flagged first, then by citations
Research works printed but not linked: 3
- 2023, Next generation sequencing
- 2024, 10x Genomics
- Eng, 2021, Plus ultra: genome-wide spatial transcriptomics with RNA seqFISH+
Cited by
Sorted by citations
Shah, S., & D’Souza, G. G. M. (2025) · Cells · doi:10.3390/cells14100732 · Cited by 66 · see citing works
“Emerging spatial transcriptomics platforms, such as MERSCOPE (Vizgen), FISSEQ, ExSeq, Slide-seq, and 10x Genomics Visium, now allow for high-resolution, multiplexed mapping of RNA molecules within intact 3D cultures [199–201].”
Beyond single biomarkers: multi-omics strategies to predict immunotherapy outcomes in blood cancers
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Applications and development of in situ nucleic acid visualization techniques
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- Authors
- Fortner, A., & Bucur, O.
- Affiliations
- Heidelberg University (DE)
- Institutul National Victor Babes (RO)
- Corresponding author
- Bucur, Octavian
- ORCID
- Bucur, Octavian
- Venue
- Frontiers in Cell and Developmental Biology
- Publisher
- Frontiers Media SA
- Published
- 2024-07-22
- Volume
- 12
- Pages
- 1378875-1378875
- ISSN
- 2296-634X (ISSN-L)
- PMC
- PMC11298486
- OpenAlex
- W4400896461
- Cited by
- 10
- References
- 94
- Citations by year
- 2026: 5 · 2025: 5
- FWCI
- 0.38
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Cell and Developmental Biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Unitatea Executiva pentru Finantarea Invatamantului Superior, a Cercetarii, Dezvoltarii si Inovarii (PN-III-P4-ID-PCE-2020-2027, PCE 153/2021 Projects of Excellence Funding in RDI, Contract No. 31PFE/30.12.2021.)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
- Keywords
- spatial transcriptomics, expansion microscopy, in situ sequencing, in situ hybridization, super-resolution imaging, RNA detection, cell type annotation, gene expression in tissues, disease mechanisms
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
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