Detail of Fig4b_tsCOX8-Kohinoor2_raw

Time-lapse observation of mitochondria labeled with tsCOX8-Kohinoor2.0 in live COS7 cells.
Time-lapse observation of mitochondria labeled with tsCOX8-Kohinoor2.0 in live COS7 cells.
Release, Updated
Image data
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Data size
326.7 MB

Cercopithecidae ( NCBITaxon:9527 )
Cell Line
COS7 cell ( NA )

Molecular Function (MF)
Biological Process (BP)
mitochondrial fusion ( GO:0008053 ) mitochondrial fission ( GO:0000266 )
Cellular Component (CC)
mitochondrial matrix ( GO:0005759 )
Biological Imaging Method
time lapse microscopy ( Fbbi:00000249 )
X scale
20 nanometer/pixel
Y scale
20 nanometer/pixel
Z scale
T scale
0.5 second per frame

Image Acquisition
Experiment type
Microscope type
Acquisition mode
Contrast method
Microscope model
Detector model
Objective model
Filter set

Summary of Methods
See details in Wazawa T, et. al. (2021) Microscopy (Oxf), Aug 9;70(4):340-352.
Related paper(s)

Tetsuichi Wazawa, Ryohei Noma, Shusaku Uto, Kazunori Sugiura, Takashi Washio, Takeharu Nagai (2021) A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging., Microscopy (Oxford, England), Volume 70, Number 4, pp. 340-352

Published in 2021 Aug 9

(Abstract) Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.
(MeSH Terms)

Takeharu Nagai , Osaka University , SANKEN , Nagai Laboratory

OMERO Dataset
OMERO Project