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Related Experiment Video

Updated: Apr 9, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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Combining fluorescence and bioluminescence microscopy.

Kazuhito Goda1, Yoko Hatta-Ohashi1, Ryutaro Akiyoshi1

  • 1Corporate Research and Development Center, Olympus Corporation, Hachioji, Tokyo, 192-8512, Japan.

Microscopy Research and Technique
|June 23, 2015
PubMed
Summary

Simultaneous bioluminescence and fluorescence microscopy enables tracking of single-cell gene expression and signaling pathways. This technique reveals heterogeneous cellular responses to stimuli, advancing live-cell imaging for biological research.

Keywords:
NF-κBPKCluciferinsingle-cell analysis

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Microscopy Techniques

Background:

  • Gene expression varies among individual cells even under identical stimuli.
  • Analyzing signal transduction and gene expression sequentially in single cells is crucial but challenging.
  • Existing methods using fluorescence and bioluminescence microscopy cannot be easily combined due to background interference.

Purpose of the Study:

  • To develop and optimize a method for simultaneous fluorescence and bioluminescence microscopy.
  • To enable sequential observation of signal transduction and gene expression in live single cells.
  • To investigate the heterogeneity of gene expression responses in relation to upstream signaling.

Main Methods:

  • Optimized optical filter sets for green and yellow fluorescent protein imaging.
  • Determined optimal luciferin concentration (>100 μM) for cell permeability and minimal background fluorescence.
  • Developed a sequential imaging protocol combining fluorescence and bioluminescence microscopy.

Main Results:

  • Successfully imaged protein kinase C alpha translocation and nuclear factor of kappa light polypeptide B activation.
  • Demonstrated that gene expression responses are not always concordant with upstream signaling.
  • Showcased the technique's ability to analyze heterogeneous gene expression and signaling in live single cells.

Conclusions:

  • The optimized dual-mode microscopy allows simultaneous observation of cellular signaling and gene expression.
  • This technique provides insights into the heterogeneity of single-cell responses to stimuli.
  • It is a powerful tool for dissecting complex molecular events in live cells.