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Updated: Jul 1, 2026

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

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Image-Based Profiling in Live Cells Using Live Cell Painting.

Thaís Moraes-Lacerda¹, Mariana Rodrigues-Da-Silva¹, Shantanu Singh²

¹Departamento de Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil.

|October 13, 2025

Summary

This summary is machine-generated.

This study introduces a cost-effective live-cell high-content analysis (HCA) method using acridine orange. This approach enables dynamic cellular profiling and drug discovery by visualizing subcellular structures in real-time.

Keywords:

Acridine orange staining Fluorescence microscopy High-content screening Image-based phenotyping Live-cell imaging

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Area of Science:

Cellular imaging and analysis
Drug discovery and toxicology
Phenotypic profiling

Background:

High-content analysis (HCA) traditionally uses fixed cells, limiting dynamic process observation.
Live-cell imaging offers expanded capabilities for studying real-time cellular responses.
Existing fixed-cell assays have limitations in detecting subtle, sublethal phenotypic changes.

Purpose of the Study:

To present a simple, cost-effective, and scalable live-cell HCA protocol.
To enable real-time phenotypic profiling and dose-response analysis.
To complement fixed-cell assays by preserving cell viability and enabling dynamic measurements.

Main Methods:

Utilized acridine orange (AO), a metachromatic fluorescent dye, for live-cell HCA.
Employed a two-channel fluorescence readout to visualize nuclei and acidic compartments.
Integrated the method with high-throughput microscopy and computational analysis.

Main Results:

Demonstrated visualization of distinct subcellular structures in live cells.
Enabled phenotypic profiling and dose-response analysis of various perturbants.
Provided a framework for detecting subtle, sublethal phenotypic changes.

Conclusions:

The AO-based live-cell HCA protocol offers a versatile platform for uncovering complex cellular phenotypes.
This method complements fixed-cell assays, enhancing drug discovery and toxicology studies.
The streamlined analysis pipeline ensures efficient and reproducible interpretation of image-based data.