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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

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Two-dimensional nonlinear structured illumination microscopy with rsEGFP2.

Shaoheng Li¹, Ryo Tamura^2,3, Kota Banzai^2,4

¹School of Electrical and Computer Engineering, University of Georgia, Athens, GA, USA.

Biomedical Optics Express

|October 20, 2025

Summary

This summary is machine-generated.

Patterned depletion nonlinear structured illumination microscopy (PD-NSIM) achieves sub-80 nm resolution in live-cell imaging using the rsEGFP2 fluorescent protein. This technique offers a promising combination of speed, resolution, and longevity for advanced biological imaging.

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Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

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Published on: July 6, 2019

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Nonlinear structured illumination microscopy (NSIM) offers high resolution but has limited published applications.
Existing NSIM techniques face challenges in speed, resolution, and fluorophore longevity.

Purpose of the Study:

To demonstrate patterned depletion NSIM (PD-NSIM) using the fluorescent protein rsEGFP2.
To achieve high-resolution live-cell imaging with improved speed and longevity.

Main Methods:

Utilized patterned depletion NSIM (PD-NSIM) with the fast-switching fluorescent protein rsEGFP2.
Performed live 2D imaging of actin filaments in U2OS cells.
Analyzed image resolution in both real-space and Fourier-space.

Main Results:

Achieved sub-80 nm resolution in live-cell imaging of actin filaments.
Demonstrated the effectiveness of rsEGFP2 as a fluorophore for PD-NSIM.
Showcased the first live 2D PD-NSIM application with rsEGFP2.

Conclusions:

PD-NSIM with rsEGFP2 is a viable method for high-resolution live-cell imaging.
rsEGFP2 provides a valuable combination of speed, resolution, and longevity for NSIM.
This technique advances the capabilities of super-resolution microscopy for biological studies.