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Related Concept Videos

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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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Related Experiment Video

Updated: Jan 10, 2026

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples
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RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution.

Ruzbeh Mosadeghi1, Daniel Foyt2, Louis Sharp3

  • 1Department of Medicine, University of California, San Francisco, San Francisco, CA, 94143, United States of America.

Biorxiv : the Preprint Server for Biology
|November 24, 2025
PubMed
Summary
This summary is machine-generated.

We developed RainBar, an optical barcoding system for live-cell imaging, enabling high-throughput functional genomics. This method tracks dynamic cellular phenotypes with single-cell resolution, advancing our understanding of biological processes.

Keywords:
CRISPRFluorescent proteinsLive-cell imagingOptical barcodingPooled screeningSingle-cell analysis

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Area of Science:

  • Functional genomics
  • Cellular imaging
  • Molecular biology

Background:

  • Existing high-throughput screening methods often miss dynamic, time-resolved cellular phenotypes due to reliance on endpoint sequencing.
  • Live-cell imaging offers potential for studying dynamic processes but lacks scalable multiplexing for pooled screens.

Purpose of the Study:

  • To develop an optical barcoding system (RainBar) for pooled live-cell imaging.
  • To enable high-throughput, single-cell resolution analysis of dynamic phenotypes in functional genomics screens.
  • To dissect signaling dynamics, such as NF-κB pathway kinetics, using this novel platform.

Main Methods:

  • RainBar system utilizing lentiviral co-delivery of spectrally distinct fluorescent proteins for multiplexed barcoding (up to 64 perturbations).
  • Advanced techniques including single-template viral production and codon diversification to enhance barcode accuracy.
  • Ratio-based spectral unmixing, inverted cytoplasmic segmentation, and a multilayer perceptron classifier for precise barcode identification.

Main Results:

  • Demonstrated accurate barcode identification in both arrayed and pooled formats.
  • Successfully applied RainBar to study NF-κB signaling dynamics, revealing stimulus-specific RelA translocation kinetics.
  • Identified known and novel regulators of NF-κB signaling in pooled CRISPRi screens, including Ino80 complex subunits and KAT2A.

Conclusions:

  • RainBar is a versatile platform for multiplexed, image-based functional genomics in live cells.
  • The system facilitates the study of dynamic signaling architectures and cellular phenotypes.
  • This technology opens new avenues for dissecting complex biological processes with high resolution and throughput.