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. 2024 Mar 20;146(11):7135-7139.
doi: 10.1021/jacs.3c13368. Epub 2024 Mar 5.

Near-Infrared Perylenecarboximide Fluorophores for Live-Cell Super-Resolution Imaging

Ze-Hua Wu  1   2 , Xingfu Zhu  1 , Qiqi Yang  1 , Yulian Zagranyarski  1 , Krishna Mishra  2 , Hilmar Strickfaden  3 , Ronald P Wong  4 , Thomas Basché  2 , Kaloian Koynov  1 , Mischa Bonn  1 , Chen Li  1 , Xiaomin Liu  1 , Klaus Müllen  1   2
Affiliations

Near-Infrared Perylenecarboximide Fluorophores for Live-Cell Super-Resolution Imaging

Ze-Hua Wu et al. J Am Chem Soc. .

Abstract

Organic near-infrared (NIR) photoblinking fluorophores are highly desirable for live-cell super-resolution imaging based on single-molecule localization microscopy (SMLM). Herein we introduce a novel small chromophore, PMIP, through the fusion of perylenecarboximide with 2,2-dimetheylpyrimidine. PMIP exhibits an emission maximum at 732 nm with a high fluorescence quantum yield of 60% in the wavelength range of 700-1000 nm and excellent photoblinking without any additives. With resorcinol-functionalized PMIP (PMIP-OH), NIR SMLM imaging of lysosomes is demonstrated for the first time in living mammalian cells under physiological conditions. Moreover, metabolically labeled nascent DNA is site-specifically detected using azido-functionalized PMIP (PMIP-N3) via click chemistry, thereby enabling the super-resolution imaging of nascent DNA in phosphate-buffered saline with a 9-fold improvement in spatial resolution. These results indicate the potential of PMIP-based NIR blinking fluorophores for biological applications of SMLM.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Synthesis of PMIP and Its Derivatives
Reagents and conditions: (i) benzylamine, triethylamine, NMP, 90 °C, 30 min, 70% for 2a; (ii) acetone, trifluoroacetic acid, 60 °C, 12 h, 75% for PMIP; (iii) BBr3, dichloromethane, 0 °C, 24 h, 60% for PMIP-OH and 85% for 5; (iv) 1,3-dibromopropane, K2CO3, DMF, rt, 24 h, 70%; (v) (3-azidopropyl)dimethylamine, acetonitrile, 60 °C, 3 h, 70%.
Figure 1
Figure 1
Photophysical properties of PMIP. (a) Absorption and emission spectrum in DMSO. (b) Typical single-molecule fluorescence time trace of PMIP. (c) On–off duty cycle of PMIP in air. (d) Histogram of detected photons per switching event and single-exponential fit of PMIP in air.
Figure 2
Figure 2
NIR live-cell SMLM imaging of lysosomes with PMIP-OH. (a) Conventional wide-field image of lysosomes labeled with PMIP-OH in living U2OS cells. (b) SMLM image of lysosomes labeled with PMIP-OH in living U2OS cells within 150 s. The four marked square areas (1, 2, 3, 4) represent four individual lysosomes. (c) Time sequence super-resolution images of lysosomes (yellow rectangle marked in b) with 30 s intervals. Scale bars: 2 μm in a and b, 200 nm in c.
Figure 3
Figure 3
Labeling nascent DNA with PMIP-N3. (a) Diagram of nascent DNA incorporating with F-ara-EdU and subsequently detected with PMIP-N3. (b) Conventional wide-field images of nascent DNA metabolically labeled with (+)/without (−) F-ara-EdU and then performing the same click chemistry with PMIP-N3, scale bars: 50 μm.
Figure 4
Figure 4
SMLM imaging of nascent DNA labeled by PMIP-N3 in PBS solution. (a, b) Reconstructed SMLM images of nascent DNA and corresponding conventional wide-field images (insets). (c) Profile of red line marked in a, white arrows indicating the position. (d) Profile of red line marked in b, white arrows indicating the position. Scale bars: 2 μm.
See this image and copyright information in PMC

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