Stimulated Emission Depletion Microscopy with Lower Laser Power
Junle Qu*, Wei Yan, Zhigang Yang, Luwei Wang, Jia Zhang, and Jialin Wang
Center for Biomedical Optics and Photonics, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, China 518060
*E-mail: jlqu@szu.edu.cn
Abstract
Stimulated emission depletion (STED) microscopy 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 achieve successful STED imaging with reduced STED laser power. The first method relies on the development of novel STED imaging techniques such as adaptive optics, phasor analysis and digital enhancement to lower the depletion power. The other 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. In addition, a dual-color STED microscope with a single laser source is developed, and spatial resolutions of 75 nm and 104 nm are obtained for mitochondria and tubulin in HeLa cells.
Reference
[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 High-Resolution 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 super-resolution microscope using a single laser source, J. Biophotonics, 13: e202000057, 2020.
Biography:
Junle Qu is a Professor in the College of Physics and Optoelectronic Engineering at Shenzhen University. He is the director of Center for Biomedical Optics and Photonics, Shenzhen University and also the director of Biomedical Photonics Committee of Chinese Optical Society. He is the Fellow of SPIE. His work is focused on biomedical optical imaging and imaging guided optical therapy. He has published more than 320 papers in peer reviewed journals and serving in the editorial boards of Biosensors, Frontiers of Optoelectronics and JIOHS.
Optical superresolution microscopy of molecular mechanisms of disease
Clemens Kaminski1*
1Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK CB3 0AS
*E-mail: cfk23@cam.ac.uk
Abstract
The self-assembly of proteins into ordered macromolecular structures is fundamental to a variety of diseases, for example in neurodegeneration, where misfolded proteins aggregate into toxic fibrillar shapes, or during virus replication, where the assembly of functional virions in the host cell is a tightly organized process.
In this talk, I will give an overview of optical imaging techniques [1-3] that allow us to gain insights into protein self-assembly reactions in vitro [4 – 7], in cells [8 – 10], and in live model organisms of disease [11]. In particular, we wish to understand how proteins nucleate to form functional or toxic structures and to correlate such information with biological phenotypes. I will show how single molecule localization microscopy, and developments in high speed structured illumination microscopy are capable of tracking the aggregation of proteins in vitro and in vivo, and how such data are interpreted in the context of disease [11-17].
References
[1] F. Stroehl and C.F. Kaminski, Optica (2016)
[2] M. Fantham and C.F. Kaminski, Nat. Phot. (2016)
[3] F. Stroehl et al., Sci. Rep. (2016)
[4] G.S. Kaminski Schierle, et al, JACS (2011)
[5] D. Pinotsi et al, Nano Letters (2013)
[6] R. Laine et al, Nat. Comms (2018)
[7] R. Laine et al, eLife (2018)
[8] E. Avezov et al., Nat. Cell Biol.(2018)
[9] D. Pinotsi et al, PNAS (2016)
[10] M. Lu et al, JBC (2019)
[11] C. Michel, et al, JBC (2014)
[12] T. Murakami, et al, Neuron (2015)
[13] H. Wong, et al, Neuron (2017)
[14] G. Fusco, et al, Nat. Comms. (2016)
[15] S. Qamar, et al, Cell (2018)
[16] J. Lautenschlaeger et al., Nat. Comms. (2018)
[17] T Shigeoka et al., Cell Reports (2019)
Biography:
Clemens Kaminski is Professor of Chemical Physics and Head of the Department of Chemical Engineering and Biotechnology at the University of Cambridge, UK. His group develops advanced photonic technologies for the study of molecular mechanisms of disease. He has published more than 200 papers, serves on numerous scientific advisory boards, and is a Fellow of the Optical Society of America.