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Related Experiment Video

Updated: Jun 11, 2026

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
10:41

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Published on: June 7, 2019

Improved sensitivity for two-photon frequency-domain lifetime measurement.

Arnold D Estrada1, Andrew K Dunn

  • 1Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, C0800, Austin, TX 78712, USA. ArnoldE@mail.utexas.edu

Optics Express
|July 1, 2010
PubMed
Summary

This study introduces a new method to enhance two-photon frequency-domain lifetime measurements, particularly in low signal-to-background scenarios. The technique utilizes modulated excitation and detects specific frequency shifts in the emission to improve sensitivity.

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Area of Science:

  • Spectroscopy and Photophysics
  • Advanced Measurement Techniques
  • Biophysical Chemistry

Background:

  • Two-photon excitation (TPE) enables deeper tissue imaging but often suffers from poor signal-to-background ratios.
  • Frequency-domain lifetime measurements offer advantages in complex biological samples.
  • Improving sensitivity in TPE lifetime measurements is crucial for advanced applications.

Purpose of the Study:

  • To develop a novel method for enhancing measurement sensitivity in two-photon frequency-domain lifetime measurements.
  • To address challenges posed by poor signal-to-background conditions in TPE spectroscopy.
  • To provide a robust mathematical model for analyzing TPE phosphorescence emission.

Main Methods:

  • Sinusoidal modulation of the two-photon excitation source.

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Last Updated: Jun 11, 2026

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
10:41

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Published on: June 7, 2019

Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging
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Advanced Compositional Analysis of Nanoparticle-polymer Composites Using Direct Fluorescence Imaging

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  • Detection of the second harmonic of the modulation frequency in the emission signal.
  • Development and validation of a mathematical model relating phase shift and demodulation to phosphorescence lifetime and modulation frequency.

    • Main Results:

    • Demonstrated improved measurement sensitivity for two-photon frequency-domain lifetime measurements.
    • Observed new frequency terms in phosphorescence emission due to the quadratic nature of TPE.
    • Validated the mathematical model by showing agreement between experimental phase shift/demodulation and theoretical predictions across all frequency components.

    Conclusions:

    • The proposed modulation technique significantly enhances sensitivity in TPE lifetime measurements under challenging conditions.
    • The developed mathematical model accurately describes the relationship between experimental parameters and phosphorescence lifetime.
    • This method offers a promising advancement for sensitive spectroscopic analysis using two-photon excitation.