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Updated: May 3, 2026

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
07:26

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Published on: October 15, 2016

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Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection

Heath E Johnson1, Jason M Haugh1

  • 1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.

Current Protocols in Cell Biology
|February 11, 2014
PubMed

Insights

This study details total internal reflection fluorescence (TIRF) microscopy and advanced image analysis for tracking signal transduction dynamics in mammalian cells. These methods enable precise characterization of phosphoinositide 3-kinase (PI3K) signaling and can be adapted for other cellular processes.

Area of Science:

  • Cellular biology
  • Microscopy techniques
  • Signal transduction pathways

Background:

  • Class I phosphoinositide 3-kinases (PI3Ks) play critical roles in cellular signaling.
  • Understanding the spatiotemporal dynamics of PI3K signaling is crucial for deciphering cellular functions.
  • Existing imaging and analysis methods may have limitations in capturing rapid dynamic events.

Purpose of the Study:

  • To present a comprehensive unit on utilizing total internal reflection fluorescence (TIRF) microscopy for studying PI3K signaling dynamics.
  • To introduce and detail advanced image analysis methods for characterizing spatiotemporal signaling events.
  • To provide protocols applicable to live-cell TIRF experiments and adaptable for other fluorescent biosensor studies.

Main Methods:

  • Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy.
  • Detailed protocols for image acquisition, processing, and segmentation.
  • Development and application of advanced image analysis techniques for spatiotemporal dynamics.

Main Results:

  • Established protocols for live-cell TIRF imaging of PI3K signaling.
  • Demonstrated advanced image analysis methods for characterizing signaling dynamics.
  • Validated the general applicability of TIRF methods for live-cell studies.

Conclusions:

  • TIRF microscopy combined with advanced image analysis provides powerful tools for studying PI3K signaling dynamics.
  • The presented methods offer a robust framework for investigating spatiotemporal cellular processes.
  • These techniques can be extended to analyze a broader range of cellular events using fluorescent biosensors.

Keywords:
cell biophysicscell migrationimage analysisimage processingsignal transduction

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