Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data

Hiroaki Ohishi; Hiroshi Ochiai

doi:10.1007/978-1-0716-4136-1_24

Image Analysis Protocol for DNA/RNA/Immunofluorescence (IF)-seqFISH Data

Methods Mol Biol. 2025:2856:419-432. doi: 10.1007/978-1-0716-4136-1_24.

Authors

Hiroaki Ohishi¹, Hiroshi Ochiai²

Affiliations

¹ Division of Gene Expression Dynamics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
² Division of Gene Expression Dynamics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. ochiai.hiroshi.403@m.kyushu-u.ac.jp.

PMID: 39283466
DOI: 10.1007/978-1-0716-4136-1_24

Abstract

Imaging-based spatial multi-omics technologies facilitate the analysis of higher-order genomic structures, gene transcription, and the localization of proteins and posttranslational modifications (PTMs) at the single-allele level, thereby enabling detailed observations of biological phenomena, including transcription machinery within cells and tissues. This chapter details the principles of such technologies, with a focus on DNA/RNA/immunofluorescence (IF) sequential fluorescence in situ hybridization (seqFISH). A comprehensive step-by-step protocol for image analysis is provided, covering image preprocessing, spot detection, and data visualization. For practical application, complete Jupyter Notebook codes are made available on GitHub ( https://github.com/Ochiai-Lab/seqFISH_analysis ).

Keywords: Higher-order genomic structure; Imaging; Transcription; seqFISH.

MeSH terms

Animals
DNA* / genetics
Fluorescent Antibody Technique* / methods
Humans
Image Processing, Computer-Assisted* / methods
In Situ Hybridization, Fluorescence* / methods
RNA* / analysis
RNA* / genetics
RNA* / metabolism
Software*

Substances

RNA
DNA