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8OPTIMIZE THE PERFORMANCE OF STIMULATED EMISSION DEPLETION (STED) NANOSCOPY
THROUGH OPTICAL METHODS AND PROBES
JUNLE QU, ZHIGANG YANG, WEI YAN, LUWEI WANG, JIA LI, JIA ZHANG, JIALIN WANG
Center for Biomedical Optics and Photonics & College of Physics and Optoelectronic Engineering, Shenzhen University, China
ABSTRACT
Stimulated emission depletion (STED) nanoscopy is an advanced super-resolution imaging technique which can provide a lateral resolution of 10-80 nm and longitudinal resolution of 30-600 nm with high imaging speed. These abilities stimulated its increasing contribution in visualizing and understanding many complex biological structures and dynamic functions at nanoscale level. However, for live cell STED imaging, the use of intense STED laser could be detrimental as it can cause severe photodamage to live cells, tissues and even fluorophores. Moreover, use of intense STED laser is likely to accelerate photobleaching process of fluorophores which may impede long-term STED imaging. We proposed two strategies to optimize STED imaging performance with reduced STED laser power. The first method relies on the development of novel STED imaging techniques such as adaptive optics, phasor analysis, digital enhancement and temporal and spatial modulation to lower the depletion power. The other method relies upon the development of new dedicated STED probes with better photostability and lower saturation intensity, including perovskite quantum dots, carbon dots, organosilicon nanohybrids and enhanced squaraine variant probe. Furthermore, a dual-color STED microscope with a single laser source is developed and demonstrated for simultaneous STED imaging of mitochondria and tubulin in HeLa cells.
Repetition rate (80MHz) c Ita«focn /stso Idomit
Time channels without increasing the depletion power
(a)
Figure: A space-time modulation method (modulated STED) based on FLIM technique
REFERENCES
[1] W. Yan, Y. Yang, Y. Tan, X. Chen, Y. Li, J. Qu, T. Ye, Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples, Photonics Res., 5:176-181(2017).
[2] L. Wang, W. Yan, R. Li, X. Weng, J. Zhang, Z. Yang, L. Liu, J. Qu, Aberration correction for improving the image quality in STED microscopy using the genetic Algorithm, Nanophotonics, 7:1971-1980(2018).
[3] Y. Chen, L. Wang, W. Yan, X. Peng, J. Qu, J. Song, Elimination of Re-excitation in Stimulated Emission Depletion Nanoscopy Based on Photon Extraction in a Phasor Plot, Laser Photonics Rev., 14: 1900352(2020).
[4] S. Ye, W. Yan, M. Zhao, X. Peng, J. Song, J. Qu, Low-Saturation-Intensity, High-Photostability, and HighResolution STED Nanoscopy Assisted by CsPbBr3 Quantum Dots, Adv. Mater., 30:201800167(2018).
[5] X. Yang, Z. Yang, Z. Wu, Y. He, C. Shan, P. Chai, C. Ma, M. Tian, J. Teng, D. Jin, W. Yan, P. Das, J. Qu, P. Xi, Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine variant probe, Nature Communications, 11, 1:1-9(2020).
[6] J. Wang, J. Zhang, L. Wang, X. Gao, Y. Shao, L. Liu, Z. Yang, W. Yan, J. Qu, Dual-color STED superresolution microscope using a single laser source, J. Biophotonics, 13: e202000057(2020).
