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Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics

Spatial biology is a rapidly growing research field that focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for the identific...

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Autores principales: Linares, Alejandro, Brighi, Carlo, Espinola, Sergio, Bacchi, Francesco, Crevenna, Álvaro H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177490/
https://www.ncbi.nlm.nih.gov/pubmed/37174710
http://dx.doi.org/10.3390/cells12091310
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author Linares, Alejandro
Brighi, Carlo
Espinola, Sergio
Bacchi, Francesco
Crevenna, Álvaro H.
author_facet Linares, Alejandro
Brighi, Carlo
Espinola, Sergio
Bacchi, Francesco
Crevenna, Álvaro H.
author_sort Linares, Alejandro
collection PubMed
description Spatial biology is a rapidly growing research field that focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for the identification of multiple targets in situ. Nonetheless, the number of genes that can be reliably visualized is limited by the diffraction of light. Here, we investigate the effect of structured illumination (SIM), a super-resolution microscopy approach, on the performance of single-gene transcript detection in spatial transcriptomics experiments. We performed direct mRNA-targeted hybridization in situ sequencing for multiple genes in mouse coronal brain tissue sections. We evaluated spot detection performance in widefield and confocal images versus those with SIM in combination with 20×, 25× and 60× objectives. In general, SIM increases the detection efficiency of gene transcript spots compared to widefield and confocal modes. For each case, the specific fold increase in localizations is dependent on gene transcript density and the numerical aperture of the objective used, which has been shown to play an important role, especially for densely clustered spots. Taken together, our results suggest that SIM has the capacity to improve spot detection and overall data quality in spatial transcriptomics.
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spelling pubmed-101774902023-05-13 Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics Linares, Alejandro Brighi, Carlo Espinola, Sergio Bacchi, Francesco Crevenna, Álvaro H. Cells Communication Spatial biology is a rapidly growing research field that focuses on the transcriptomic or proteomic profiling of single cells within tissues with preserved spatial information. Imaging-based spatial transcriptomics uses epifluorescence microscopy, which has shown remarkable results for the identification of multiple targets in situ. Nonetheless, the number of genes that can be reliably visualized is limited by the diffraction of light. Here, we investigate the effect of structured illumination (SIM), a super-resolution microscopy approach, on the performance of single-gene transcript detection in spatial transcriptomics experiments. We performed direct mRNA-targeted hybridization in situ sequencing for multiple genes in mouse coronal brain tissue sections. We evaluated spot detection performance in widefield and confocal images versus those with SIM in combination with 20×, 25× and 60× objectives. In general, SIM increases the detection efficiency of gene transcript spots compared to widefield and confocal modes. For each case, the specific fold increase in localizations is dependent on gene transcript density and the numerical aperture of the objective used, which has been shown to play an important role, especially for densely clustered spots. Taken together, our results suggest that SIM has the capacity to improve spot detection and overall data quality in spatial transcriptomics. MDPI 2023-05-04 /pmc/articles/PMC10177490/ /pubmed/37174710 http://dx.doi.org/10.3390/cells12091310 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Linares, Alejandro
Brighi, Carlo
Espinola, Sergio
Bacchi, Francesco
Crevenna, Álvaro H.
Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title_full Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title_fullStr Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title_full_unstemmed Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title_short Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics
title_sort structured illumination microscopy improves spot detection performance in spatial transcriptomics
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10177490/
https://www.ncbi.nlm.nih.gov/pubmed/37174710
http://dx.doi.org/10.3390/cells12091310
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