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Updated: May 10, 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

Encoded multisite two-photon microscopy.

Mathieu Ducros¹, Yannick Goulam Houssen, Jonathan Bradley

¹Institut National de la Santé et de la Recherche Médicale U603, Paris 75006, France.

Proceedings of the National Academy of Sciences of the United States of America

|June 27, 2013

Summary

This summary is machine-generated.

Encoded multisite two-photon microscopy (eMS2PM) enables faster, simultaneous brain activity imaging. This scanless method overcomes scanning speed limitations for studying neuronal networks in vivo.

Keywords:

multiplexing multipoint scanless two-photon microscopy voltage-sensitive dyes

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Last Updated: May 10, 2026

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Area of Science:

Neuroscience
Optical Imaging
Biophysics

Background:

Scanning two-photon microscopy (2PM) is crucial for noninvasive brain activity investigation.
A key limitation of 2PM is its scanning speed, hindering real-time neuronal network analysis.
High-speed, simultaneous imaging is needed to capture action potential-like activity.

Purpose of the Study:

To introduce and validate encoded multisite two-photon microscopy (eMS2PM) as a solution to 2PM's speed limitations.
To demonstrate eMS2PM's capability for simultaneous, high-temporal-resolution imaging of multiple targets in depth.
To enable optical interrogation of neuronal network dynamics.

Main Methods:

Developed eMS2PM, a scanless imaging technique utilizing a liquid crystal spatial light modulator and a digital micromirror device.
Subbeams are encoded with unique binary amplitude modulation sequences for site-specific identification.
Simultaneous fluorescence signal collection and site-specific decoding onto a single photodetector.

Main Results:

Successfully imaged spike-like voltage transients in cultured cells.
Demonstrated in vivo imaging of fluorescence transients, including neuronal calcium signals and red blood cell dynamics in cortical capillaries.
Achieved simultaneous acquisition of activity from multiple sites in depth.

Conclusions:

eMS2PM overcomes the speed limitations of conventional 2PM.
This method allows for simultaneous, high-resolution imaging of neuronal network activity in vivo.
eMS2PM is a powerful new tool for neuroscience research.