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Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

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Protocol for quantifying phase-separated condensates in living cells using HILO microscopy imaging and genetic

Axel Espinoza¹, Steven Ingersoll², Xiaojun Ren³

¹Department of Integrative Biology, University of Colorado Denver, Denver, CO 80217-3364, USA.

|October 18, 2025

Summary

This study introduces a new method to measure how epigenetic factors cluster via phase separation in living cells. The protocol uses advanced microscopy and genetic tools to visualize and quantify this crucial process for genome organization.

Keywords:

Biophysics CRISPR cell Biology microscopy

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Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

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Published on: January 5, 2024

Area of Science:

Cell biology
Molecular biology
Genetics

Background:

Epigenetic factors play a key role in gene regulation and genome organization.
Spatial clustering of these factors through phase separation is an emerging mechanism.
Quantifying this phase separation capacity in living cells is essential for understanding its biological significance.

Purpose of the Study:

To present a novel protocol for quantifying the phase separation capacity of epigenetic factors in living cells.
To enable detailed analysis of epigenetic factor behavior and its impact on genome organization.

Main Methods:

Utilizing highly inclined and laminated optical sheet (HILO) microscopy for high-resolution imaging.
Employing CRISPR-Cas9 gene editing to generate a specialized cell line.
Incorporating CRE-LoxP or dTAG-inducible degradation systems for controlled manipulation of fusion proteins.
Developing imaging and analysis procedures to quantify phase separation.

Main Results:

The protocol successfully allows for the visualization and quantification of epigenetic factor phase separation in real-time.
The generated cell line enables precise control over fusion protein levels and their subsequent phase separation behavior.

Conclusions:

This protocol provides a robust method for studying the biophysical properties of epigenetic factors.
It opens new avenues for investigating the role of phase separation in transcriptional regulation and maintaining genome architecture.