Spatiotemporal resolution of light-controlled imaging technology has played a huge role in life science research. Stimulated Raman scattering (SRS) microscopy has made great progress in recent years as a vibrational imaging technique due to its rich molecular information, chemical bond labeling, and multi-channel imaging. However, multi-channel reversible light-controlled SRS imaging is still not possible
Recently, Fanghao Hu's research group in the Department of Chemistry of Tsinghua University has developed a reversible light-switched multi-channel SRS imaging technology, which has obtained a series of light-switched Raman molecules with excellent properties by coupling asymmetric diaryl ethylene (DAE) and polyalkyne carbon rainbow (Carbow), which undergo significant vibrational frequency changes and signal enhancement under visible light irradiation, which are used for high-spatiotemporal selective multi-channel imaging in living cells, providing a new method for studying complex life processes and subcellular interactions.
Figure 1. Asymmetric DAE-polyyne structure (a) and its excellent photoswitching properties: UV-Vis absorption spectrum (b), optical switching conversion (c), optical switching fatigue stability (d), SRS frequency change (e), SRS signal enhancement (f).
By introducing asymmetric heterocycles, DAE-polyyne molecules are able to undergo a reversible cyclization reaction under visible light irradiation, with the closed-loop maximum absorption wavelength redshifted to the near-infrared region (Figure 1). Moreover, the asymmetric DAE-polyyne compound exhibits efficient optical switching conversion (up to 90%) and excellent anti-fatigue stability, with stable signals after dozens of cycles of illumination. At the same time, the asymmetric DAE-polyyne exhibits different Raman scattering frequencies in the open-loop and closed-loop states, with frequency variations of up to 24 cm-1, which lays the foundation for the SRS detection of reversible optical switches. Finally, due to the near-infrared absorption of DAE-polyyne in the closed-loop state, the electronic pre-resonance effect can be generated, and the SRS signal intensity is significantly higher than that in the open-loop state.
Figure 2. Carbow-switch molecular structure and 16-channel photoswitch Raman frequency
Based on the excellent optical switching properties of asymmetric DAE-polyynes, the authors accurately adjusted the Raman frequency of DAE-polyyne by changing its end-capped substituents, triple bond number, and isotope labeling, and obtained a series of reversible photocontrolled probes with 16 different switching vibration frequencies, which were named "Carbow-switch" (Fig. 2). The Carbow-switch produces significant frequency changes and signal enhancement under visible illumination, demonstrating its potential for multi-channel, highly specific optical-switched SRS imaging.
Figure 3. Live-cell multichannel light-controlled SRS imaging to study subcellular interactions in oxidative stress and protein phase separation.
The authors further explored live-cell imaging with Carbow-switch, enabling multichannel light-controlled SRS imaging of different organelle targets, including mitochondria, lysosomes, and cell membranes. Carbow-switch maintains excellent biocompatibility and photoswitching performance in living cells. Finally, the authors also achieved highly spatiotemporally selective, light-controlled multichannel SRS imaging in live cells. By labeling specific organelles with Carbow-switch and imaging using time-series SRS, the authors tracked dynamic processes such as mitochondrial aggregation in living cells under oxidative stress, lysosomal migration between adjacent cells, and lysosomal interactions during intracellular protein phase separation (Figure 3). The highly spatiotemporally selective photocontrolled multi-channel SRS imaging technology provides a new means to study multi-component cellular processes.
In this study, we developed a multi-channel reversible light-switched SRS imaging technology, which skillfully coupled the polyalkyne carbon rainbow with the asymmetric diaryl ethylene structure, and obtained 16 light-controlled Raman frequencies located in the cell silent interval by co-adjusting the molecular absorption spectrum and vibrational spectra, and realized the reversible light-switched multi-channel SRS imaging with high spatiotemporal selection in living cells through organelle targeting, and selectively tracked the interaction of organelles in the process of oxidative stress, cell-to-cell transport and protein phase separation. The development of this technique provides a powerful tool for studying spatiotemporally resolved dynamic processes and subcellular interactions in complex cellular environments.
The research results were recently published in Nature Communications, and the first author of the paper is Yang Yueli, a doctoral student, and Bai Xueyang, a doctoral student, has made important contributions to this work, and the corresponding author is Associate Professor Hu Fanghao.
Paper Information:
Photoswitchable polyynes for multiplexed stimulated Raman scattering microscopy with reversible light control
Yueli Yang, Xueyang Bai and Fanghao Hu
Nat. Commun., 2024, 15, 2758. DOI: 10.1038/s41467-024-46904-6