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Super-resolution Fluorescence Microscopy

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Updated: May 21, 2026

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Published on: August 29, 2025

Pulse-shaping multiphoton FRET microscopy.

Meredith H Brenner¹, Dawen Cai, Sarah R Nichols

¹Applied Physics Program, University of Michigan, 450 Church Street, Ann Arbor, MI, USA 48109.

Proceedings of Spie--The International Society for Optical Engineering

|June 28, 2012

Summary

This summary is machine-generated.

This study introduces a new two-photon Fluorescence Resonance Energy Transfer (FRET) microscopy method using pulse-shaping for selective fluorophore excitation in live cells. This technique enables precise FRET measurements, advancing quantitative biophysical studies.

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Area of Science:

Biophysics
Microscopy
Molecular Biology

Background:

Fluorescence Resonance Energy Transfer (FRET) microscopy is vital for studying biomolecular interactions and cellular processes.
Spectral overlap in conventional FRET presents challenges for quantitative analysis.
Selective excitation of fluorophores is key for accurate FRET stoichiometry.

Purpose of the Study:

To develop a novel two-photon FRET microscopy technique for selective fluorophore excitation.
To overcome limitations of conventional femtosecond lasers in two-photon FRET.
To enable quantitative FRET studies in live cells with improved accuracy.

Main Methods:

Utilized pulse-shaping via multiphoton intrapulse interference for tailored excitation pulses.
Implemented rapid switching between selective donor and acceptor fluorophore excitation.
Applied the technique to live cells expressing fluorescent proteins mCerulean and mCherry.

Main Results:

Demonstrated selective excitation of fluorophores using pulse-shaping in two-photon FRET.
Successfully detected two-photon FRET in live cells with high precision.
Overcame the bandwidth and tuning limitations of conventional femtosecond lasers.

Conclusions:

The developed pulse-shaping technique enables selective excitation for two-photon FRET.
This method paves the way for advanced two-photon FRET stoichiometry in live-cell imaging.
Offers a powerful new tool for quantitative biophysical investigations.