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Super-resolution Fluorescence Microscopy

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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

Multimodal two-photon imaging using a second harmonic generation-specific dye.

Mutsuo Nuriya^1,2, Shun Fukushima³, Atsuya Momotake³

¹Department of Pharmacology School of Medicine, Keio University, 35 Shinanomachi, Tokyo 160-8582, Japan.

Nature Communications

|May 10, 2016

Summary

This summary is machine-generated.

Researchers developed Ap3, a novel non-fluorescent dye for second harmonic generation (SHG) imaging. This membrane potential-sensitive dye enables clear SHG signals without fluorescence, advancing multimodal imaging in biological research.

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Area of Science:

Biophysics
Chemical Biology
Neuroscience

Background:

Second harmonic generation (SHG) imaging offers unique visualization of biological processes.
Current SHG dyes suffer from strong fluorescence, limiting applications and causing signal overlap.
Existing fluorescent dyes like FM4-64 produce both SHG and fluorescence, complicating data interpretation.

Purpose of the Study:

To develop a novel, non-fluorescent dye for SHG imaging.
To create a membrane potential-sensitive dye for neuronal activity monitoring.
To enable true multimodal two-photon imaging by separating SHG and fluorescence signals.

Main Methods:

Synthesis and characterization of a new organic dye, Ap3.
Testing Ap3's SHG and fluorescence properties in biological samples.
Evaluating Ap3's performance in neurons, including membrane potential sensitivity and response to action potentials.
Comparing Ap3 with the conventional dye FM4-64 regarding signal specificity, photostability, and photodamage.

Main Results:

Ap3 is the first non-fluorescent, membrane potential-sensitive SHG-active organic dye.
Ap3 generates strong SH signals at the plasma membrane with negligible fluorescence.
Ap3 demonstrates linear membrane potential sensitivity and rapid responses to neuronal action potentials.
Ap3 exhibits superior photostability and reduced photodamage compared to FM4-64.
The SHG-specificity of Ap3 allows independent simultaneous imaging of SHG and various fluorescence reporters.

Conclusions:

Ap3 is a breakthrough dye for SHG imaging, overcoming limitations of fluorescent dyes.
Ap3 enables true multimodal two-photon imaging by providing distinct SHG and fluorescence channels.
This dye significantly enhances the capability for visualizing complex biological events in real-time.