image: Fig. 1 Two-color mSTED super-resolution imaging
Credit: OEA
A new publication from Opto-Electronic Advances; DOI 10.29026/oea.2024.240035 discusses multiplexed stimulated emission depletion nanoscopy for multi-color live-cell long-term imaging.
In the field of cell biology, an increasing number of studies are focusing on the intricate network of interactions among subcellular structures. As a powerful imaging tool, super-resolution fluorescence microscopy broke the diffraction limit, enabling biologists to observe subcellular structures with nanoscale resolution. Among super-resolution fluorescence microscopy, stimulated emission depletion microscopy (STED) is one of the leading techniques beyond the diffraction limit, and it ensures minimal artifacts by its immediate super-resolution microscopic properties without post-processing.
In the last decade, the need to study the interactions between subcellular structures has led to an increasing interest and application of multicolor live-cell STED, most conventionally achieved by using multiple excitation-depletion beam pairs. However, increasing the number of depletion beams not only makes the system more complex and dramatically increases the construction cost, but also increases the likelihood of photo-bleaching and more severe photo-cytotoxicity, which is not conducive to live-cell imaging. On the other hand, the use of a single depletion beam along with multiple excitation beams limits the range of available excitation wavelengths, and dividing the restricted band into multiple densely arranged spectral channels and reducing crosstalk between them poses a significant challenge. Currently, this approach is typically limited to two- or three-color imaging.
Consequently, researchers have employed fluorescence lifetime information to achieve multicolor imaging. Fluorescence lifetime is the average time a fluorescent molecule spends in the excited state and can be utilized to distinguish between different fluorescent molecules. However, multicolor STED based on fluorescence lifetimes is currently only applicable to fixed cells due to the difficulty of (i) screening out live-cell fluorescent markers with brightness and anti-bleaching properties suitable for STED imaging, (ii) simultaneously labeling multiple subcellular structures in living cells, and (iii) separating different fluorescent probes in the same spectral channel using appropriate analytical method. Currently, biologists face a lack of effective methods for studying the dynamics and function of subcellular structures using STED microscopy.
Based on the above challenges, the authors of this article have developed multiplexed stimulated emission depletion nanoscopy (mSTED), which allows for simultaneous observation of more structures with limited photobleaching and phototoxicity. The researchers screened a series of suitable fluorescent probe combinations capable of labeling multiple subcellular structures simultaneously. These live-cell fluorescent probes with similar spectral identity were subsequently separated by a phasor analysi. mSTED achieved 5-color live-cell STED imaging and revealed long-term interactions between different subcellular structures. The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.
The researchers first tested the performance of mSTED by two-color imaging (Fig. 1), which showed that it could successfully separate different subcellular structures and caused only 4% crosstalk. Compared to confocal microscopy, the resolution of mSTED was significantly improved (~60 nm), enabling the observation of subcellular structures in much finer detail.
To verify the performance of mSTED in photo-bleaching and photo-cytotoxicity, the researchers conducted a comparative analysis with mSTED and conventional multicolor STED methods (Fig. 2). Two depletion lasers were required in conventional two-color STED imaging. After 11 minutes of imaging, the fluorescence signal of the microtubules decreased to 13.4% of the initial value, and the mitochondria transformed into swollen and round shapes, indicating severe photo-bleaching and photo-cytotoxicity. In contrast, mSTED required only one depletion laser. After 11 minutes of imaging, the fluorescence signal of the microtubules was still 31.5% and the shape of the mitochondria did not exhibit significant changes.
Researchers employed mSTED for live-cell long-term imaging, enabling the simultaneous observation of five subcellular structures (Fig.3). The large enough information of long-term multi-color mSTED allowed us to discover interesting phenomena in cell biology. As an illustration, microtubules hold up some space beneath the nucleus for other organelles to move around, and mitochondrial fission and fusion happened in this limited space under the cooperation of ER and microtubules. These results highlight the superiority of multi-color mSTED in long-term live-cell imaging of multiple structures, enabling the systematical interpretation of organelle interaction network at the same time.
Keywords: optical nanoscopy / phasor analysis / multicolor live cell imaging
# # # # # #
Cuifang Kuang is a professor at the College of Optical Science and Engineering, Zhejiang University, and deputy director of the State Key Laboratory of Extreme Photonics and Instrumentation, mainly engaged in the research of super-resolution microscopic imaging and super-resolution lithography. He presides over various projects such as the National Outstanding Youth Fund and the Major Instrumentation Special Fund of the National Natural Science Foundation of China. He has published more than 180 related papers as the first or corresponding author, and more than 100 invention patents. 2019 Wang Daheng Optics Award - Optics Award for Young and Middle-aged Scientists and Technologists. The results won the second prize of China Technological Invention in 2019 (2/6), the first prize of China Optical Science and Technology Award in 2018 (2/6), the third prize of Zhejiang Scientific and Technological Advancement in 2020 (2/7), Innovation Achievement Award of Zhejiang Province Industry-University-Research Cooperation in 2023 (1/10). Personal homepage: https://person.zju.edu.cn/cfkuang
Yubing Han is a Distinguished Researcher at the College of Optical Science and Engineering, Zhejiang University, and received her B.S. and Ph.D. degrees from Huazhong University of Science and Technology in 2013 and 2018, respectively. Her main research interests are in super-resolution microscopic imaging technology and its biological applications. She presided over one youth fund of the National Natural Science Foundation of China (NSFC), and participated in one key research and development program of the Ministry of Science and Technology (MOST) and one major research program of the National Natural Science Foundation of China (NSFC) as a sub-topic leader. She has published more than ten papers as first/corresponding author in Nature Communications (2017, SCI citation 140+), Cell Reports Methods (2023, Cover Article) and other journals. She serves as a guest editor for Photonics and other SCI journals. Personal homepage: https://person.zju.edu.cn/0618248
# # # # # #
Opto-Electronic Advances (OEA) is a rapidly growing high-impact, open access, peer reviewed monthly SCI journal with an impact factor of 15.3 (Journal Citation Reports for IF2023). OEA has been indexed in SCI, EI, DOAJ, Scopus, CA and ICI databases, and expanded its Editorial Board to 31 members from 17 countries with an average h-index of 62.
The journal is published by The Institute of Optics and Electronics, Chinese Academy of Sciences, aiming at providing a platform for researchers, academicians, professionals, practitioners, and students to impart and share knowledge in the form of high quality empirical and theoretical research papers covering the topics of optics, photonics and optoelectronics.
# # # # # #
More information: http://www.oejournal.org/oea
Editorial Board: http://www.oejournal.org/oea/editorialboard/list
All issues available in the online archive (http://www.oejournal.org/oea/archive).
Submissions to OEA may be made using ScholarOne (https://mc03.manuscriptcentral.com/oea).
ISSN: 2096-4579
CN: 51-1781/TN
Contact Us: oea@ioe.ac.cn
Twitter: @OptoElectronAdv (https://twitter.com/OptoElectronAdv?lang=en)
WeChat: OE_Journal
# # # # # #
Huang YR, Zhang ZM, Tao WL et al. Multiplexed stimulated emission depletion nanoscopy (mSTED) for 5-color live-cell long-term imaging of organelle interactome. Opto-Electron Adv 7, 240035 (2024). doi: 10.29026/oea.2024.240035
Journal
Opto-Electronic Advances