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Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy
08:39

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Published on: January 7, 2019

Pulsed interleaved excitation fluctuation imaging.

Jelle Hendrix1, Waldemar Schrimpf, Matthias Höller

  • 1Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany. jelle_hendrix@hotmail.com.

Biophysical Journal
|August 27, 2013
PubMed
Summary
This summary is machine-generated.

Pulsed interleaved excitation with fluctuation imaging (PIE-FI) enhances protein studies in living cells. This method accurately measures protein dynamics, interactions, and concentrations, offering powerful quantitative biophysical insights.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Spectroscopy

Background:

  • Fluorescence fluctuation imaging (FI) is crucial for studying protein dynamics and interactions within living cells.
  • Pulsed interleaved excitation (PIE) enables precise, simultaneous measurement of fluorescence intensity and lifetime for multiple fluorophores.

Purpose of the Study:

  • To combine PIE with FI methods (PIE-FI) for advanced quantitative analysis of protein behavior.
  • To validate PIE-FI techniques including raster image correlation spectroscopy (RICS), number and brightness analysis (N&B), and lifetime-based correlation spectroscopy (RLICS).

Main Methods:

  • Implemented PIE-RICS for diffusion and molecular brightness measurements of Venus fluorescent protein.
  • Applied dead-time corrected PIE-N&B to study eGFP oligomerization and maturation.
  • Utilized PIE-RLICS for quantitative concentration determination, FRET analysis, and resolving codiffusing species.

Main Results:

  • Demonstrated accurate diffusion and brightness measurements with PIE-RICS, outperforming single-point PIE-FCS.
  • Showcased cross-talk-free dual-color PIE-RICS for comparing fluorescent protein pairs.
  • Achieved sensitive concentration measurements down to <30 pM using PIE-RLICS.
  • Enabled robust dual-color FRET analysis and resolved FRET species in cells.

Conclusions:

  • PIE-FI offers enhanced capabilities for quantitative biophysical studies.
  • The integration of PIE with RICS, N&B, and lifetime analysis provides powerful tools for investigating protein dynamics, interactions, and concentrations in complex biological systems.