Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength

Abstract

Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.

Description
Keywords
Dronpa, fluorescent dye, Kohinoor, Padron, protein, reversibly photoswitchable fluorescent protein, rsEGFP, Skylan NS, Skylan S, unclassified drug, photoprotein, Article, controlled study, fluorescence, fluorescence microscopy, human, human cell, illumination, imaging, information processing, microscope, protein degradation, structured illumination microscopy, fluorescence microscopy, HeLa cell line, light, metabolism, nonlinear system, procedures, HeLa Cells, Humans, Light, Luminescent Proteins, Microscopy, Fluorescence, Nonlinear Dynamics
Citation
Lu-Walther, H.-W., Hou, W., Kielhorn, M., Arai, Y., Nagai, T., Kessels, M. M., et al. (2016). Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength. 11(10). https://doi.org//10.1371/journal.pone.0165148
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License
CC BY 4.0 Unported