Journal: Nature Communications
Article Title: In-depth single molecule localization microscopy using adaptive optics and single objective light-sheet microscopy
doi: 10.1038/s41467-025-62198-8
Figure Lengend Snippet: A Schematic of the soSMARt acquisition pipeline, leveraging on dedicated SMARt microfabricated devices containing 45° mirrors and embedded fiduciary markers surrounding the sample. It allows (1) in-depth optical aberration correction using Adaptive Optics, (2) PSF calibration for soSPIM-based 3D single-molecule localization, (3) real-time mechanical drift correction in 3D using SMARtrack, and (4) precise extended-volume reconstruction. B Optical setup composed of an inverted microscope, the soSPIM beam steering unit, a deformable mirror (DM) for aberration correction, and a XYZ piezo stage for mechanical drift correction. C Image plane of the lamin B1 protein in a COS-7 cell suspended in a SMARt device well. The well containing the cell (dashed line) is surrounded by numerous fluorescent fiduciary markers (four examples shown in white boxes). D Mean residual drift after real-time feedback-loop registration using SMARtrack, computed from the localization precision of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n=5$$\end{document} n = 5 fiduciary markers in the polymer of a SMARt device ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{mean}}}\pm {{\rm{s}}}.{{\rm{e}}}.{{\rm{m}}}$$\end{document} mean ± s . e . m ). E In-depth astigmatism-based 3D SMLM of the Lamin B1 nuclear envelope of a COS-7 cell reconstructed from a single plane DNA-PAINT acquisition at 13.5 µm above the coverslip. Colors indicate the z distance from the coverslip. Left: SMLM reconstructions at three intermediate depths. Inset shows an example of an astigmatism-based DNA-PAINT image (scale bars are 5 µm).
Article Snippet: The soSPIM beam steering unit and AO systems were mounted onto an inverted two stages Nikon Ti2 (Microscope) equipped with a XY motorized stage (Nikon TI2-S-SE-E), an epi-fluorescence excitation path (IntensityLight C-HGFIE, Nikon) on the lower stage with GFP, CY3 and Cy5 filter sets (GFP-3035d, Cy3-4040c, and Cy5-4040c from Semrock respectively), a white light transmission path (D-LH/LC Arc lamp, Nikon), and a XYZ piezo stage (Nano-LPS200, Mad City Lab).
Techniques: Inverted Microscopy, Polymer
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![A Schematic of the soSMARt acquisition pipeline, leveraging on dedicated SMARt microfabricated devices containing 45° mirrors and embedded fiduciary markers surrounding the sample. It allows (1) in-depth optical aberration correction using Adaptive Optics, (2) PSF calibration for soSPIM-based 3D single-molecule localization, (3) real-time mechanical drift correction in 3D using SMARtrack, and (4) precise extended-volume reconstruction. B Optical setup composed of an inverted microscope, the soSPIM beam steering unit, a deformable mirror (DM) for aberration correction, and a <t>XYZ</t> <t>piezo</t> stage for mechanical drift correction. C Image plane of the lamin B1 protein in a COS-7 cell suspended in a SMARt device well. The well containing the cell (dashed line) is surrounded by numerous fluorescent fiduciary markers (four examples shown in white boxes). D Mean residual drift after real-time feedback-loop registration using SMARtrack, computed from the localization precision of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n=5$$\end{document} n = 5 fiduciary markers in the polymer of a SMARt device ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{mean}}}\pm {{\rm{s}}}.{{\rm{e}}}.{{\rm{m}}}$$\end{document} mean ± s . e . m ). E In-depth astigmatism-based 3D SMLM of the Lamin B1 nuclear envelope of a COS-7 cell reconstructed from a single plane DNA-PAINT acquisition at 13.5 µm above the coverslip. Colors indicate the z distance from the coverslip. Left: SMLM reconstructions at three intermediate depths. Inset shows an example of an astigmatism-based DNA-PAINT image (scale bars are 5 µm).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0799/pmc12460799/pmc12460799__41467_2025_62198_Fig1_HTML.jpg)