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Updated: Jul 30, 2025

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

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Published on: April 24, 2019

Structured Illumination Microscopy Improves Spot Detection Performance in Spatial Transcriptomics.

Alejandro Linares¹, Carlo Brighi², Sergio Espinola¹

¹Epigenetics and Neurobiology Unit, European Molecular Biology Laboratory, 00015 Rome, Italy.

|May 13, 2023

Summary

This summary is machine-generated.

Structured illumination microscopy (SIM) enhances gene transcript spot detection in spatial transcriptomics. This super-resolution technique improves data quality by increasing the efficiency of visualizing single-gene transcripts within tissues.

Keywords:

deconvolution microscopy gene expression in situ sequencing spatial transcriptomics structured illumination super-resolution

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Area of Science:

Molecular Biology
Microscopy
Genomics

Background:

Spatial biology analyzes cellular and molecular data within tissue context.
Imaging-based spatial transcriptomics uses microscopy for in situ gene expression analysis.
Light diffraction limits the resolution and detection of genes in traditional microscopy.

Purpose of the Study:

To evaluate the impact of structured illumination microscopy (SIM) on single-gene transcript detection in spatial transcriptomics.
To compare SIM performance against widefield and confocal microscopy for transcript localization.
To determine how objective magnification and spot density influence SIM's effectiveness.

Main Methods:

Direct mRNA-targeted hybridization in situ sequencing was employed.
Experiments were conducted on mouse coronal brain tissue sections.
Spot detection was assessed using widefield, confocal, and SIM imaging with 20×, 25×, and 60× objectives.

Main Results:

SIM generally increased the detection efficiency of gene transcript spots compared to widefield and confocal modes.
The fold increase in detected spots varied with gene transcript density and objective numerical aperture.
Higher numerical apertures were particularly beneficial for resolving densely clustered transcripts.

Conclusions:

Structured illumination microscopy significantly improves spot detection in spatial transcriptomics.
SIM enhances the overall data quality for transcriptomic profiling in situ.
SIM offers a valuable approach to overcome resolution limitations in spatial transcriptomics.