Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction

Tetsuichi Wazawa, Yoshiyuki Arai, Yoshinobu Kawahara, Hiroki Takauchi, Takashi Washio, Takeharu Nagai

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80nm with illumination power densities as low as ~1W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of superresolution fluorescence microscopy for live-cell imaging.

Original languageEnglish
Pages (from-to)89-98
Number of pages10
JournalMicroscopy
Volume67
Issue number2
DOIs
Publication statusPublished - Apr 1 2018

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Computer-Assisted Image Processing
Optical Imaging
demodulation
image reconstruction
Demodulation
Image reconstruction
Fluorescence
Lighting
Polarization
proteins
Proteins
Imaging techniques
fluorescence
Fluorescence microscopy
polarization
cells
Fluorescence Microscopy
excitation
illumination
microscopy

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Instrumentation
  • Radiology Nuclear Medicine and imaging

Cite this

Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction. / Wazawa, Tetsuichi; Arai, Yoshiyuki; Kawahara, Yoshinobu; Takauchi, Hiroki; Washio, Takashi; Nagai, Takeharu.

In: Microscopy, Vol. 67, No. 2, 01.04.2018, p. 89-98.

Research output: Contribution to journalArticle

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