Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing

Luwei Wang; Yue Chen; Jiaqing Guo; Xiaoyu Weng; Wei Yan; Jun Song; Tong Ye; Junle Qu

doi:10.1038/s41377-024-01711-y

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Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing

Original Articles

- Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing
- Light: Science & Applications Vol. 14, Issue 1, Pages: 258-270(2025)
- Affiliations：
  
  1.Center for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen, 518060, China
  2.The Photonics Center of Shenzhen University, Shenzhen University, Shenzhen, 518060, China
  3.Department of Bioengineering, CU-MUSC Bioengineering Program, Clemson University, Charleston, South Carolina, 29634, USA
- Author bio：
  
  Jun Song (songjun@szu.edu.cn)
  Tong Ye (ye7@clemson.edu)
  Junle Qu (jlqu@szu.edu.cn)
- Funds：
- DOI：10.1038/s41377-024-01711-y
  CLC：
- Published：31 January 2025，
  
  Published Online：03 January 2025，
  
  Received：16 May 2024，
  
  Revised：13 November 2024，
  
  Accepted：2024-12-03
- Accepted：
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Wang, L. W. et al. Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing. Light: Science & Applications, 14, 258-270 (2025).
DOI：

Wang, L. W. et al. Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing. Light: Science & Applications, 14, 258-270 (2025). DOI： 10.1038/s41377-024-01711-y.

摘要

Abstract

Multicolor microscopy and super-resolution optical microscopy are two widely used techniques that greatly enhance the ability to distinguish and resolve structures in cellular imaging. These methods have individually transformed cellular imaging by allowing detailed visualization of cellular and subcellular structures

as well as organelle interactions. However

integrating multicolor and super-resolution microscopy into a single method remains challenging due to issues like spectral overlap

crosstalk

photobleaching

phototoxicity

and technical complexity. These challenges arise from the conflicting requirements of using different fluorophores for multicolor labeling and fluorophores with specific properties for super-resolution imaging. We propose a novel multicolor super-resolution imaging method called phasor-based fluorescence spatiotemporal modulation (Phasor-FSTM). This method uses time-resolved detection to acquire spatiotemporal data from encoded photons

employs phasor analysis to simultaneously separate multiple components

and applies fluorescence modulation to create super-resolution images. Phasor-FSTM enables the identification of multiple structural components with greater spatial accuracy on an enhanced laser scanning confocal microscope using a single-wavelength laser. To demonstrate the capabilities of Phasor-FSTM

we performed two-color to four-color super-resolution imaging at a resolution of ~λ/5 and observed the interactions of organelles in live cells during continuous imaging for a duration of over 20 min. Our method stands out for its simplicity and adaptability

seamlessly fitting into existing laser scanning microscopes without requiring multiple laser lines for excitation

which also provides a new avenue for other super-resolution imaging technologies based on different principles to build multi-color imaging systems with the requirement of a lower budget.

关键词

Keywords

references

Lobingier, B. T. et al. An approach to spatiotemporally resolve protein interaction networks in living cells. Cell 169 , 350–360. e12 (2017)..

Norregaard, K. et al. Manipulation and motion of organelles a nd single molecules in living cells. Chem. Rev. 117 , 4342–4375 (2017)..

Huang, J. W. et al. The relevance of organelle interactions in cellular senescence. Theranostics 12 , 2445–2464 (2022)..

Bückers, J. et al. Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses. Opt. Express 19 , 3130–3143 (2011)..

Wang, J. R. et al. Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection. Cell Rep. Methods 2 , 100170 (2022)..

Brelje, T. C. et al. Multicolor laser scanning confocal immunofluorescence microscopy: practical application and limitations. Methods Cell Biol. 70 , 165–244 (2002). 245e-249e..

Hershko, E. et al. Multicolor localization microscopy and point-spread-function engineering by deep learning. Opt. Express 27 , 6158–6183 (2019)..

Wu, Y. C. et al. Multiview confocal super-resolution microscopy. Nature 600 , 279–284 (2021)..

Sigal, Y. M., Zhou, R. B. & Zhuang, X. W. Visualizing and discovering cellular structures with super-resolution microscopy. Science 361 , 880–887 (2018)..

Zhang, Y. D. et al. Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging. Nat. Methods 17 , 225–231 (2020)..

Schermelleh, L. et al. Super-resolution microscopy demystified. Nat. Cell Biol. 21 , 72–84 (2019)..

Jin, D. Y. et al. Nanoparticles for super-resolution microscopy and single-molecule tracking. Nat. Methods 15 , 415–423 (2018)..

Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19 , 780–782 (1994)..

Klar, T. A. et al. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97 , 8206–8210 (2000)..

Blom, H. & Widengren, J. Stimulated emission depletion microscopy. Chem. Rev. 117 , 7377–7427 (2017)..

Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198 , 82–87 (2000)..

Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102 , 13081–13086 (2005)..

Heintzmann, R. & Huser, T. Super-resolution structured illumination microscopy. Chem. Rev. 117 , 13890–13908 (2017)..

Sauer, M. & Heilemann, M. Single-molecule localization microscopy in eukaryotes. Chem. Rev. 117 , 7478–7509 (2017)..

Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Prim. 1 , 39 (2021)..

Hugelier, S., Colosi, P. L. & Lakadamyali, M. Quantitative single-molecule localization microscopy. Annu. Rev. Biophys. 52 , 139–160 (2023)..

Balzarotti, F. et a l. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355 , 606–612 (2017)..

Eilers, Y. et al. MINFLUX monitors rapid molecular jumps with superior spatiotemporal resolution. Proc. Natl Acad. Sci. USA 115 , 6117–6122 (2018)..

Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17 , 217–224 (2020)..

Rickert, J.D. et al. 4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter. Proc. Natl Acad. Sci. USA 121 , e2318870121 (2024)..

Kuang, C. F. et al. Breaking the diffraction barrier using fluorescence emission difference microscopy. Sci. Rep. 3 , 1441 (2013)..

Prigent, S. et al. Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on an array detector. Opt. Lett. 48 , 498–501 (2023)..

Korobchevskaya, K. et al. Intensity weighted subtraction microscopy approach for image contrast and resolution en hancement. Sci. Rep. 6 , 25816 (2016)..

Qiu, Y. X. et al. Resolution and contrast enhancement in weighted subtraction microscopy by deep learning. Opt. Lasers Eng. 164 , 107503 (2023)..

Wang, L. W. et al. Implementation of a fluorescence spatiotemporal modulation super-resolution microscope. Opt. Lett. 47 , 581–584 (2022)..

Weber, G. Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements. J. Phys. Chem. 85 , 949–953 (1981)..

Jameson, D. M., Gratton, E. & Hall, R. D. The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry. Appl. Spectrosc. Rev. 20 , 55–106 (1984)..

Ranjit, S., Malacrida, L. & Gratton, E. Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card. Microsc. Res. Tech. 81 , 980–989 (2018)..

Chaudhry, A., Shi, R. & Luciani, D. S. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells. Am. J. Physiol. -Endocrinol. Metab. 318 , E87–E101 (2020)..

Valente, A. J. et al. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture. Acta Histochem. 119 , 315–326 (2017)..

Elbaz-Alon, Y. et al. A dynamic interface between vacuoles and mitochondria in yeast. Dev. Cell 30 , 95–102 (2014)..

Wong, Y. C., Ysselstein, D. & Krainc, D. Mitochondria-lysosome contacts regulate mitochondrial fission via RAB7 GTP hydrolysis. Nature 554 , 382–386 (2018)..

Wong, Y. C. et al. Regulation and function of mitochondria–lysosome membrane contact sites in cellular homeostasis. Trends Cell Biol. 29 , 500–513 (2019)..

Guo, Y. T. et al. Visualizing intracellular organelle and cytoskeletal interactions at nanoscale resolution on millisecond timescales. Cell 175 , 1430–1442. e17 (2018)..

Wang, C. et al. Dynamic tubulation of mitochondria drives mitochondrial network formation. Cell Res. 25 , 1108–1120 (2015)..

Guo, J. J. et al. Conformational details of quantum dot-DNA resolved by Förster resonance energy transfer lifetime nanoruler. ACS Nano 13 , 505–514 (2019)..

Li, H. et al. Multi-functional carbon dots for visualizing and modulating ROS-induced mitophagy in living cells. Adv. Funct. Mater. 33 , 212141 (2023)..

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