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Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics

Authors :
Alejandro Linares
Carlo Brighi
Sergio Espinola
Francesco Bacchi
Álvaro H. Crevenna
Source :
Cells. 12:1310
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

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.

Subjects

Subjects :
biophysics
General Medicine

Details

ISSN :
20734409
Volume :
12
Database :
OpenAIRE
Journal :
Cells
Accession number :
edsair.doi.dedup.....1f35fb85804330cd1428addf67265a35
Full Text :
https://doi.org/10.3390/cells12091310