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Related Experiment Video

Updated: May 20, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Multicolor two-photon tissue imaging by wavelength mixing.

Pierre Mahou¹, Maxwell Zimmerley, Karine Loulier

¹Laboratory for Optics and Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique UMR 7645 and INSERM U696, Palaiseau, France.

|July 10, 2012

Summary

This study demonstrates simultaneous multicolor imaging using synchronized laser pulses to excite three distinct chromophores. This advanced technique enables detailed visualization of biological structures like brain tissue and developing embryos.

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Area of Science:

Biophysics
Microscopy
Optical Imaging

Background:

Multicolor imaging is crucial for visualizing complex biological systems.
Simultaneous excitation of multiple fluorophores presents significant technical challenges.

Purpose of the Study:

To develop a method for simultaneous two-photon excitation of three distinct chromophores.
To enable advanced multicolor imaging of biological samples.

Main Methods:

Utilized synchronized pulses from a femtosecond laser and an optical parametric oscillator.
Achieved spatiotemporal overlap of laser beams for enhanced excitation.
Employed distinct chromophores with varied absorption spectra.

Main Results:

Demonstrated simultaneous two-photon excitation of three chromophores.
Achieved submicrometer overlay of color channels for precise imaging.
Successfully performed volume and live multicolor imaging of 'Brainbow'-labeled tissues.
Reported simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

Conclusions:

The developed method allows for efficient and simultaneous multicolor excitation.
This technique significantly advances the capabilities of multiphoton microscopy for biological research.
Opens new avenues for high-resolution imaging of dynamic biological processes.