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17PROGRESS ON STIMULATED EMISSION DEPLETION MICROSCOPY
JUNLE QU, WEI YAN, ZHIGANG YANG, LUWEI WANG, JIA ZHANG AND JIALIN WANG
Center for Biomedical Optics and Photonics (CBOP) College of Physics and Optoelectronic Engineering Shenzhen University
Abstract
Stimulated emission depletion (STED) microscopy, as an advanced imaging technique, was theoretically proposed in 1994 and experimentally performed later. In principle, two laser beams are needed to carry out super resolution imaging using STED method. One pulsed laser beam is used to transfer fluorescent dyes to the excited states, generating a fluorescent spot owing to optical diffraction; and the second one (STED laser) with larger pulse width, actually producing a doughnut-shape spot, is employed to selectively deactivate the fluorescent dyes lying in the overlapping region of excitation and STED laser spots. Typically, the doughnut-shaped STED beam selectively suppresses fluorescence photon emission in the periphery of the excitation beam to preferably ensure almost zero-intensity at the center. Undoubtedly, in STED, the depletion laser power is an important factor to determine the imaging resolution.
In this advanced optical microscopic technique, especially for the biological samples, selecting apposite fluorescent dyes with excellent nonlinear response is another determining factor to achieving better spatial resolution. Basically, all the fluorophores around the focal excitation spot need to be in their fluorescent 'off' state to attain an exceptionally high resolution, since the stimulated emission rate has a non-linear dependence on the intensity of the STED beam. To retrieve the high resolution images, the focal spot is then scanned across the object. Theoretically, the full-width-at-half maximum (FWHM) of the PSF at the excitation focal spot can be compressed by increasing the intensity of the STED pulses as presented in equation (1).
j1+Imax/Is'
where, Ar is the lateral resolution, A represents the FWHM of the diffraction limited PSF, Imax is the peak intensity of the STED laser, and Isstands for the threshold intensity needed in order to achieve saturated emission depletion.
The STED microscopy can provide a lateral resolution of 10-80 nm and a longitudinal resolution of 30-600 nm with high imaging speed. These abilities of the STED microscopy stimulated its increasing contribution in visualizing and understanding many complex biological structures and dynamic functions in a plethora of cell and tissue types at nanoscale level. However, for live cell STED imaging, the use of intense laser could be detrimental as it can cause severe photodamage to the live cells, tissues and even the used fluorophores. Moreover, use of intense STED laser beam is likely to accelerate the photobleaching process of fluorophores which may impede the long-term STED imaging. Therefore, optimizing the STED laser power is crucial to achieve a successful good quality STED image.
Mito-635 Alexa Flour 488 Overlay
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Figure 1. (a) Principle ofDE-STED. (b) DE-STED imaging of microtubes in fixedBSC-1 cells. (c) Dual color STED imaging of cells with a single super-continuum laser. (d) STED imaging of mitochondrial dynamics with an enhanced squaraine variant probe. (e) Working principle of accurate photon extraction in the phasor plot. (f) Re-excitation eliminating STED imaging in a biological cell
In this talk I will present our recent work in STED microscopy. We proposed two major 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 STED [1, 2], phasor analysis STED [3, 4] and digitally enhanced STED [5] to lower the depletion power during the acquisition of STED images. The other significant method to minimize the STED laser power basically relies upon the development of new dedicated STED probes/fluorophores with better photostability and lower saturation intensity (/S). We developed perovskite quantum dots [6], carbon dots [7] organosilicon nanohybrids [8] and enhanced squaraine variant probe [9] for STED imaging with very lowSTED laser power. Typically, these new fluorescent materials contain superior photostability and much lower saturation intensity (1S) compared to the traditional STED probes. In addition, a dual-color STED microscope with a single laser source is developed, and the spatial resolutions of 75nm and 104 nm have been achieved for mitochondria and tubulin in HeLa cells [10].
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] L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, J. Qu, Resolution improvement in STED superresolution microscopy at low power using a phasor plot approach, Nanoscale, 10:16252-16260, 2018.
[4] 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.
[5] L. Wang, Y. Chen, X. Peng, J. Zhang, J. Wang, L. Liu, Z. Yang, W. Yan, J. Qu, Ultralow power demand in fluorescence nanoscopy with digitally enhanced stimulated emission depletion, Nanophotonics, DOI: 10.1515/nanoph-2019-0475.
[6] 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.
[7] H. Li, S. Ye, J. Guo, H. Wang, W. Yan, J. Song, J. Qu, Biocompatible carbon dots with low-saturation-intensity and high photobleaching-resistance for STED nanoscopy imaging of the nucleolus and tunneling nanotubes in living cells, Nano Research, 12: 3075-3084, 2019.
[8] L. Liang, W. Yan, X. Qin, X. Peng, H. Feng, Y. Wang, Z. Zhu, L. Liu, Y. Han, Q. Xu, J. Qu, X. Liu, Designing Sub-2 nm Organosilica Nanohybrids for Far-Field Super- Resolution Imaging , Angew. Chem., 7: 746-751, 2020.
[9] 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.
[10] 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.
