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Hkust achieves high resolution and low power fast Raman imaging of living cells

2022/8/24 Viewed：

Professor Zachary J. Smith's team from the School of Engineering Science at the University of Science and Technology of China and Professor Gao Tingjuan's team from the School of Chemistry at Central China Normal University have made new progress in the field of Raman bioimaging, proposing a rapid bioimaging method based on the combination of line scanning Raman imaging system and azo enhanced Raman probe. High resolution and low power images of organelle dynamic processes are realized. The relevant research results were published in the "High-resolution low-power hyperspectral line-scan imaging of fast cellular dynamics using" on August 15, 2022 "azo-enhanced Raman scattering probes" was published online in the prestigious academic Journal of the American Chemical Society.

Raman imaging is a labeling free single-cell analysis technique, which can obtain cell structure and composition information from molecular level, and is widely used in biomedical research. However, the Raman scattering cross section is very small, usually requires several hours of high laser illumination to obtain a frame of cell Raman image, can not capture the space-time evolution of the organelle information. Coherent Raman imaging technology can greatly improve the Raman signal, but this method can only obtain narrow band Raman spectral information, requires strong laser power, and the equipment is expensive. Raman probe, as another method of Raman signal enhancement, has the characteristics of cell permeability, targeting, low toxicity, etc., but the common alkyne labeled Raman probe can not meet the high resolution of fast cell dynamic imaging. Therefore, we designed a dynamic azo enhanced Raman imaging system, which can realize high resolution and low power fast Raman imaging of organelle dynamic processes. The comparison of traditional point-scan spontaneous Raman imaging, coherent Raman imaging and dynamic azo-enhanced Raman imaging is shown in Figure 1.

FIG. 1. Comparison of the principles of three Raman bioimaging techniques, including traditional point-scan spontaneous Raman imaging, coherent Raman imaging and dynamic azo-enhanced Raman imaging.

In this work, a new type of ultra-sensitive resonant Raman probe, namely azo-enhanced Raman scattering probe, is used, which can greatly improve the Raman signal and suppress the fluorescence background. The azo-enhanced Raman probes showed a 3-4 order of magnitude improvement in relative Raman strength compared to conventional 5-acetylidene-2-deoxyuridine (EdU) (Figure 2a). Combined with our self-designed line scan spontaneous Raman imaging system, rapid Raman imaging of multiple organelles in living cells labeled with azo-enhanced Raman probes can be achieved, and full Raman spectrum (500-3200 cm-1) information can be obtained. At low laser illumination (75 µW/µm2), the imaging rate of dynamic azo-enhanced Raman imaging system can reach up to 3.5 seconds/frame, and the confocal spatial resolution is 270 nm.

Figure 2. (a) The relative Raman strengths of multiple Raman markers; (b) Simultaneous imaging of mitochondria, lysosomes, and lipid droplets based on azo-enhanced Raman probes.

The dynamic azo-enhanced Raman imaging system used multiple Raman probes to simultaneously label the cells, achieving synchronous imaging of mitochondria, lysosomes and lipid droplets (Figure 2b). Rapid images of mitochondria and lysosomes in living cells were successfully obtained (Figure 3). Based on the image data of lysosome, the biophysical dynamic properties of lysosome movement were analyzed. The dynamic process of mitochondrial decoupling was studied by adding the uncoupling agent FCCP. The dynamic azo enhanced Raman imaging system can provide quantitative and multivariate imaging of the dynamic process of organelles, which will provide a powerful help for the research of biomedicine.

Figure 3. Rapid imaging of mitochondria and lysosomes in living cells using dynamic azo enhanced Raman imaging system.

Yu Yajun, Special Associate Researcher, School of Engineering Science, China University of Science and Technology, and Dr. Tang Yuchen, School of Chemistry, Central China Normal University, are the co-first authors of this paper. Professor Zachary J. Smith, China University of Science and Technology, and Professor Gao Tingjuan, Central China Normal University, are the co-corresponding authors of this paper. This research work has been supported by the National Natural Science Foundation of China, Anhui Provincial Science and Technology Major Project, China Postdoctoral Science Foundation and other projects.

Paper link:https://pubs.acs.org/doi/full/10.1021/jacs.2c06275

(School of Engineering Science, Department of Scientific Research)

Source: HKUST News Network

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