Chongfan Technology
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19
2025
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03
Light | Unlocking New Skills in Multicolor Super-Resolution Imaging
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Introduction
Multicolor microscopy and super-resolution optical microscopy, as two core technologies in the field of cell imaging, have greatly enhanced our ability to distinguish and resolve subcellular structures and visualize their interactions with high precision. These technologies allow for clear visualization of the complex interactions between cells, subcellular structures, and organelles, revolutionizing the research paradigm of cell imaging. However, integrating multicolor imaging with super-resolution imaging into a single system still faces many challenges, such as spectral overlap, signal crosstalk, photobleaching, phototoxicity, and technical complexity.
Recently,Shenzhen UniversityProfessor Qu Junle's teamwith the title "Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing"published their latest research findings inLight: Science & ApplicationsPhasor-FSTMa novel multicolor super-resolution imaging scheme. This scheme cleverly integrates fluorescence modulation and lifetime multiplexing technologies, using time-resolved detection methods to accurately capture the spatiotemporal information of fluorescence photons. Using advanced phasor analysis methods, the research team successfully separated multiple signal components simultaneously and generated super-resolution images using fluorescence modulation technology. It is worth mentioning that the Phasor-FSTM scheme is an improvement on the basis of the traditional confocal microscopy system, and only a single wavelength laser is needed to achieve high-precision identification of multiple organelle structures. This method is not only simple to operate but also highly adaptable, and can be easily integrated into existing laser scanning microscopy systems without relying on multi-wavelength laser excitation and multiple detectors. In addition, this technology also provides new ideas for other super-resolution imaging technologies, helping to build low-cost, high-efficiency multicolor imaging systems and pathways.
In the extraordinary journey of exploring the mysteries of life, scientists have always cherished a dream: to gain a more refined perspective and more brilliant colors to delve into the intricate structures and ever-changing dynamic processes within cells. Imagine, if we could, like magical magicians, break through the inherent limitations of microscopes and clearly witness the exquisite interactions between every tiny organelle within the cell, what an exhilarating sight it would be!
To realize this wish, we need to use the power of multicolor microscopy and super-resolution microscopy. However, traditionally, these two technologies are separate, and many difficulties have been encountered when attempting to perfectly combine them. For example, the spectra of different fluorescent dyes are prone to overlap, resulting in a significant reduction in image clarity; the long-time, high-energy laser irradiation required for super-resolution imaging may damage cells, or even induce toxic reactions; in addition, the high cost of these cutting-edge technologies has also deterred many laboratories, making them unattainable. These difficulties are like insurmountable walls, hindering scientists' efforts to explore the microscopic world of cells.
Fortunately, scientists have never stopped exploring. Recently, a team of scientists from China successfully found a new key—the Phasor-FSTM technology based on fluorescence spatiotemporal modulation and lifetime multiplexing. This technology is like opening a new window, providing an unprecedentedly clear perspective for revealing the microscopic world of cells, allowing scientists to gain a deeper insight into the mysteries within cells.
So, what is so magical about Phasor-FSTM technology? Simply put, this technology, with the clever use of a single-wavelength laser, can simultaneously possess the capabilities of multicolor imaging and super-resolution imaging. This means that scientists no longer need to use complex and expensive multi-laser systems to clearly capture multiple structures inside cells and accurately distinguish the subtle differences between them. This breakthrough not only greatly simplifies the composition of the imaging system and reduces research costs, but also opens up a whole new horizon for biological research. So, how exactly does this technology work? And what new possibilities can it bring to biological research? Next, let's unveil the mystery of Phasor-FSTM technology and explore together how it has become a new window for revealing the microscopic world of cells.
The workflow of Phasor-FSTM includes the collection and analysis of FLIM data, and data analysis is divided into three steps:
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1. Separate the FLIM data by time channel to obtain two datasets of complete fluorescence decay excited by Gaussian or donut laser beams;
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2. Perform phase analysis on the two datasets to separate each component according to lifetime;
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3. Remove the diffraction-limited signal from the Gaussian photons to achieve super-resolution, as shown inFigure 1. Through the above steps, the Phasor-FSTM technology successfully combines multicolor imaging with super-resolution imaging.
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Figure 1: Working principle of Phasor-FSTM
To demonstrate the capabilities of Phasor-FSTM, researchers performed two-color to four-color super-resolution imaging at a resolution of approximately λ/5, and continuously observed the interaction of organelles in living cells for more than 20 minutes, observing processes such as mitochondrial autophagy and the fusion of mitochondria-derived vesicles with lysosomes, providing new insights into the complex dynamics of subcellular organization and function, as shown inFigure 2. This method stands out for its simplicity and adaptability, and can be seamlessly integrated into existing laser scanning microscopes without the need for multiple laser lines for excitation. In addition, the innovative idea based on fluorescence modulation and lifetime multiplexing also provides new inspiration and ideas for other super-resolution imaging technologies based on different principles, enabling them to build multicolor imaging systems with a lower budget.
Figure 2: Long-term Phasor-FSTM imaging of living cells monitoring the interaction process between mitochondria and lysosomes
As the technology continues to be optimized and upgraded, its application range is expected to be further expanded. For example, by improving the resolution, this technology will be able to reveal more subtle structures inside cells; combined with artificial intelligence for data processing, it will be able to more efficiently analyze complex biological information; at the same time, this technology is also expected to be applied to non-fluorescent labeling imaging and in vivo diagnosis, bringing unprecedented revolutionary changes to biological and medical research.
Paper Information
Wang, L., Chen, Y., Guo, J., 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, 32 (2025).
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