CC BY
6DOI 10.24412/cl-37136-2023-1-140-142
ENHANCING STIMULATED EMISSION DEPLETION IMAGING THROUGH OPTICAL METHODS, PROBES AND DEEP-LEARNING
JUNLE QU, JIA LI, WEI YAN, LUWEI WANG, ZHIGANG YANG
College of Physics and Optoelectronic Engineering, Shenzhen University
ABSTRACT
Stimulated emission depletion (STED) microscopy is an advanced imaging technique that was first proposed in 1994 and later experimentally performed. This technique requires two laser beams to achieve superresolution imaging. The first laser beam is pulsed and is used to transfer fluorescent dyes to excited states, generating a fluorescent spot due to optical diffraction. The second laser beam, called the STED laser, has a larger pulse width and produces a doughnut-shaped spot that selectively deactivates fluorescent dyes lying in the overlapping region of the excitation and STED laser spots. The doughnut-shaped STED beam suppresses fluorescence photon emission in the periphery of the excitation beam, ensuring almost zero intensity at the center, and thus determining the imaging resolution.
To achieve better spatial resolution in STED microscopy, it is important to select appropriate fluorescent dyes with excellent nonlinear response, especially for biological samples. All the fluorophores around the focal excitation spot need to be in their fluorescent "off" state to attain exceptionally high resolution since the stimulated emission rate has a nonlinear dependence on the intensity of the STED beam. The high-resolution images are obtained by scanning the focal spot across the object. Increasing the intensity of the STED pulses can theoretically compress the full-width-at-half maximum (FWHM) of the point spread function (PSF) at the excitation focal spot, as presented in equation (1).
Ar = , A == Место для формулы. (1)
•Jt+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 Is stands for the threshold intensity needed in order to achieve saturated emission depletion.
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 Is stands for the threshold intensity needed in order to achieve saturated emission depletion.
STED microscopy is a powerful imaging technique that can achieve lateral resolutions of 10-80 nm and longitudinal resolutions of 30-600 nm with high imaging speed. These capabilities have led to its increasing use in visualizing and understanding complex biological structures and dynamic functions at the nanoscale level in a wide range of cell and tissue types. However, when used for live cell imaging, the intense laser required for STED microscopy can cause significant photodamage to cells, tissues, and fluorophores. Additionally, the use of an intense STED laser beam can accelerate the photobleaching process of fluorophores, which can impede long-term STED imaging. Therefore, optimizing the STED laser power is crucial to achieving high-quality STED images while minimizing photodamage and photobleaching.
(b)
Figure: (a) Scheme of fluorescence spatiotemporal modulation (FSTM) nanoscopy. (b) FSTM imaging of
nuclear pore complex (NPC).
In this presentation, I will discuss our recent advances in STED microscopy and related work. Our research has focused on two major strategies to achieve successful STED imaging with reduced STED laser power. The first approach involves the development of novel STED imaging techniques, such as adaptive optics STED, phasor analysis STED, and digitally enhanced STED. These techniques have been designed to lower the depletion power required during STED image acquisition.
The second significant method to minimize STED laser power involves the development of new dedicated STED probes and fluorophores with improved photostability and lower saturation intensity (IS). Our team has successfully developed several new fluorescent materials, including perovskite quantum dots, carbon dots, organosilicon nanohybrids, and enhanced squaraine variant probes. These materials enable STED imaging with very low STED laser power, exhibit superior photostability, and much lower saturation intensity compared to traditional STED probes.
Furthermore, we have developed a dual-color STED microscope with a single laser source, which has achieved spatial resolutions of 75 nm and 104 nm for mitochondria and tubulin in HeLa cells. These advancements in STED microscopy hold great promise for improving our understanding of complex biological structures and functions at the nanoscale level while minimizing the risk of photodamage and photobleaching.
In addition, we have made significant progress by developing a deep-learning-based algorithm for the generation of super-resolution images directly from diffraction-limited confocal images.
REFERENCES
[1] W. Yan, Z. 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 L., 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 superresolution microscope using a single laser source, J. Biophotonics, 13: e202000057(2020).
[7] L. Wang, J. Li, Y. Chen, Y. Yong, Z. Yang, X. Weng, W. Yan, J. Qu, Implementation of a fluorescence spatiotemporal modulation super-resolution microscope, Optics Letters,47(3):581-584(2022).
[8] B. Huang, J. Li, B. Yao, Z. Yang, E. Y. Lam, J. Zhang, W. Yan, J. Qu, Enhancing image resolution of confocal fluorescence microscopy with deep learning, PhotoniX, 4:2: doi.org/10.1186/s43074-022-00077-x (2023).
