Positive-Type Reversibly Photoswitching Red Fluorescent Protein for Dual-Color Superresolution Imaging with Single Light Exposure for Off-Switching

被引：3

作者：

Ozaki-Noma, Ryohei ^{[1
,2
]}

Wazawa, Tetsuichi ^{[1
]}

Kakizuka, Taishi ^{[1
,3
]}

Shidara, Hisashi ^{[4
]}

Takemoto, Kiwamu ^{[4
]}

Nagai, Takeharu ^{[1
,2
,3
,5
]}

机构：

[1] Osaka Univ, Inst Sci & Ind Res, SANKEN, Osaka, Ibaraki 5670047, Japan

[2] Osaka Univ, Grad Sch Frontier Biosci, Suita, Osaka 5650871, Japan

[3] Osaka Univ, Inst Open & Transdisciplinary Res Initiat, Transdimens Life Imaging Div, Suita, Osaka 5650871, Japan

[4] Mie Univ, Grad Sch Med, Tsu, Mie 5148507, Japan

[5] Hokkaido Univ, Res Inst Elect Sci, Sapporo, Hokkaido 0010020, Japan

来源：

ACS NANO | 2025年 / 19卷 / 07期

基金：

日本科学技术振兴机构; 日本学术振兴会;

关键词：

fluorescence; positive-type reversibly photoswitchingfluorescent protein; red fluorescent protein; chromophorephenolate; multiple equilibria; superresolutionimaging; nanoscopy; DISCRIMINATIVE DIMENSIONS; RESOLFT NANOSCOPY; MICROSCOPY; GENERATION; GREEN; MODULATION; KINETICS; MUTANTS;

D O I：

10.1021/acsnano.4c16847

中图分类号：

O6 [化学];

学科分类号：

0703 ;

摘要：

Positive-type reversibly photoswitching fluorescent proteins (p-rsFPs) transition to a bright on-state upon light exposure for fluorescence excitation and to a dark off-state under a different wavelength. p-rsFPs are widely used in superresolution (SR) imaging techniques, offering simplified observation procedure and enhanced biocompatibility. Although some green p-rsFPs possess adequate photoproperties for SR imaging, all red p-rsFPs (p-rsRFPs) to date exhibit suboptimal properties, limiting the color palette for multiplexed SR imaging. Here, we present a p-rsRFP, rsZACRO, with 3.0-fold brighter fluorescence, 5.3-fold faster off-switching, and 1.5-fold higher on/off contrast than rsCherry, a conventional representative p-rsRFP. Using rsZACRO with superresolution polarization demodulation/on-state polarization angle narrowing (SPoD-OnSPAN), we successfully demonstrated SR imaging in the red spectrum and dual-color SR imaging with a single light for off-switching, visualizing vimentin intermediate filaments and actin filaments at higher spatial resolution than the diffraction limit of light in a living mammalian cell.

引用

页码：7188 / 7201

页数：14

共 63 条

[1] Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting [J].