Raster-scanning Donut simplifies MINFLUX and provides alternative implement on other scanning-based microscopes

Xu et al. Light: Science & Applications (2022)11:293 https://doi.org/10.1038/s41377-022-00983-6 NEWS & VIEWS Official journal of the CIOMP 2047-7538 www.nature.com/lsa Open Access Raster-scanning Donut simplifies MINFLUX and provides alternative implement on other scanningbased microscopes Xinzhu Xu1,2,3, Shu Jia 2 and Peng Xi 1,3,4 ✉ 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Abstract A donut excitation moves around a single molecule with a zigzag configuration lattice by lattice. Such a method implemented in scanning fluorescence microscopy simplifies the conventional MINFLUX process. Consisting of hollow zero-intensity excitation, single-pixel detection, time-correlated single photon counting, and drift stabilization, the system achieves localization precision and resolution very close to conventional MINFLUX theoretically and experimentally. An averaged high-SNR reference, and pixel-registered intensity from a single molecule is essential to reconstruct localization in maximum likelihood estimation. With performance reaching nearly conventional MINFLUX’s, the proposed raster-scanning MINFLUX can inspire researchers expertized in STED or confocal setup to quickly transform to MINFLUX and develop for further exploring on bio-specimens or optical applications. The advent of super-resolution fluorescence microscopy has opened up a promising avenue to the complete understanding of cell biology, from confocal fluorescence microscopy1, which promotes signal-to-noise ratio and thus resolution, to STED2 that precisely coincides with Gaussian excitation and donut depletion, and nonlinear SIM (N-SIM)3 inspired by the excited saturation contributing to Fourier domain extension or even PALM4 /STORM5 based on single-molecule localization. They have favored researchers to directly observe the subcellular organelle morphology (mitochondrial cristae6), the fine subcellular structure (microtubules7,8, neurons skeleton9 and active zone10), and further boosted the qualitative leap in the understanding of complex life science process (heterogeneity and dynamics of membranes11 and even the first-observed enlarged fusion pores during vesicle exocytosis12). Correspondence: Peng Xi () 1 Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing 100871, China 2 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA Full list of author information is available at the end of the article In the recent lustrum, taking advantage of the characteristic of the central zero-intensity of donut excitation and the statistical estimation, Balzarotti developed MINFLUX13 in 2017, and Gwosch published 3D-MINFLUX14 in 2020. These techniques have boosted the localization precision of optical fluorescence microscopy to ~1 nm, with the corresponding spatial resolution of ~1–3 nm and temporal resolution of ~50 μs. However, the methods that achieve such ultra-precise localization precision and resolution require complicated instruments such as ultrafast optical scanning devices and delicate FPGA circuits, which inevitably leads to a significant increase in system complexity, technical barriers, and instrumentation cost. This has been a bottleneck for other laboratories to continue to develop and innovate based on the MINFLUX system, thus posing a challenge for its broad applicability. Building a more straightforward alternative system with comparable performance is one of the issues that need attention in the super-resolution fluorescence microscopy field. In 2021, the pulsed MINFLUX15 invented by Luciano et al. promised a turning point to this problem. The excitation, consisting of four parallelly spaced and equal-interval pulsed lasers, is built on an existing point- © The Author(s) 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Xu et al. Light: Science & Applications (2022)11:293 scanning confocal microscopy system. In combination with MINFLUX’s four-point targeted coordinate pattern (TCP) excitation, scanning and time-correlated singlephoton detection module, they achieved a localization precision of 1~2 nm under 1000–2000 photon counts and successfully demonstrated imaging of DNA origami samples with a feature distance of 12 nm. On this basis, MINFLUX fluorescence lifetime imaging is extended and realized by resolving a single fluosphere life signal from moving piezostage successively to configure a 7 nm-sidesquare. To further reduce the system complexity, Luciano published RASTMIN recently16, which further simplifies the system and improves the multiplexing ability with other scanning-based microscopes. Here, an innovative single-molecule localization with sequential structured illumination17 (SML-SSI) method has recently been proposed. Based upon an inverted point-scanning confocal microscope, 200 ps-pulsed-640 nm excitation is modulated into a donut after the vortex-phase plate and a quarter-wave plate. The beam then passes through the lateral scanning part with dual-axis scanning on the sample plane with nm-level accuracy. The combined upand-down drift-corrected paths (Fig. 1(a) gray part) then meet with excitation before the objective through a dichroic mirror (DM2). The lateral scan controls the donut excitation to illuminate the pre-determined square sample area (L ≈ 100 nm), which is close to TCP in a Page 2 of 5 MINFLUX and divided into K×K grid points. Scanning grid-by-grid is performed, whose structure seems like a raster, thus called RASTMIN (single-molecule localization by RASTer scanning a MINimum of light). The fluorescent signal obtained by traversing each grid is descanned by the xy-scanner, then enters the single-photon detector, and is ultimately analyzed by the time-correlated single-photon counter module. Each grid corresponds to a certain number of photon counts, forming an intensity image of K×K pixels. The acquired data is first screened for single-molecule emission on-state signals using the background threshold defined in the analytical model. Then, they compare it with a high signal-to-noise ratio image averaged by scanning a single fluorescent bead (...truncated)