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Updated: Mar 13, 2026
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Published on: December 9, 2013
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Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength
Hui-Wen Lu-Walther1, Wenya Hou2, Martin Kielhorn1
1Leibniz Institute of Photonic Technology, Jena, Germany.
Plos One
|October 27, 2016
Summary
We enhanced resolution in fluorescence microscopy using nonlinear structured illumination microscopy (NL-SIM) with the Kohinoor fluorophore. This novel approach offers improved imaging beyond traditional linear methods.
Area of Science:
- Optics and Photonics
- Biophysics
- Microscopy
Background:
- Structured illumination microscopy (SIM) offers improved resolution beyond the Abbe limit.
- Linear SIM (LSIM) is a standard technique for enhanced fluorescence imaging.
Purpose of the Study:
- To achieve further resolution enhancement in fluorescence microscopy.
- To demonstrate a novel nonlinear structured illumination microscopy (NL-SIM) technique.
Main Methods:
- Developed a two-beam nonlinear structured illumination microscope with a minor system modification.
- Utilized the photoswitching properties of the Kohinoor fluorophore for nonlinear response.
Main Results:
- Achieved resolution improvement beyond the two-fold limit of LSIM.
- Demonstrated effective use of Kohinoor's positive contrast photoswitching.
- Observed minimal degradation of Kohinoor in fixed cells over multiple switching cycles.
Conclusions:
- NL-SIM with Kohinoor offers enhanced resolution in fluorescence microscopy.
- Kohinoor is a suitable fluorophore for NL-SIM due to its photoswitching characteristics and photostability.
- This technique provides a valuable advancement for high-resolution biological imaging.
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