Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images

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Abstract

This paper presents an experimental assessment and analysis of super-resolution microscopy based on multiple-point spread function fitting of spectrally demultiplexed images using a designed DNA structure as a test target. For the purpose, a DNA structure was designed to have binding sites at a certain interval that is smaller than the diffraction limit. The structure was labeled with several types of quantum dots (QDs) to acquire their spatial information as spectrally encoded images. The obtained images are analyzed with a point spread function multifitting algorithm to determine the QD locations that indicate the binding site positions. The experimental results show that the labeled locations can be observed beyond the diffraction-limited resolution using three-colored fluorescence images that were obtained with a confocal fluorescence microscope. Numerical simulations show that labeling with eight types of QDs enables the positions aligned at 27.2-nm pitches on the DNA structure to be resolved with high accuracy.

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Acknowledgements

This research was supported by JSPS KAKENHI Grant-in-Aid for Young Scientists (B) 16K16408, a Joint Research Grant from the National Institutes of Natural Sciences (NINS), and Hikari-Mirai Young Scientist Grant 2016 of Optical Society of Japan.

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Correspondence to Takahiro Nishimura.

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Nishimura, T., Kimura, H., Ogura, Y. et al. Experimental assessment and analysis of super-resolution in fluorescence microscopy based on multiple-point spread function fitting of spectrally demultiplexed images. Opt Rev 25, 384–390 (2018). https://doi.org/10.1007/s10043-017-0379-y

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Keywords

  • Fluorescence imaging
  • Super-resolution
  • Fluorescence
  • Fluorescence coding
  • Multiple-PSF fitting