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1. The nanoscale organization of the Wnt signaling integrator Dishevelled in the vegetal cortex domain of an egg and early embryo

   https://doi.org/10.1371/journal.pone.0248197  

   Henson, John H. ; Samasa, Bakary ; Shuster, Charles B. ; Wikramanayake, Athula H. ( May 2021 , PLOS ONE)

   Klymkowsky, Michael (Ed.)

   Canonical Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos. 

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2. Improved resolution in 3D structured illumination microscopy using 3D model-based restoration with positivity-constraint

   https://doi.org/10.1364/BOE.442066  

   Van, Cong T. S. ; Preza, Chrysanthe ( November 2021 , Biomedical Optics Express)

   The performance of structured illumination microscopy (SIM) systems depends on the computational method used to process the raw data. In this paper, we present a regularized three-dimensional (3D) model-based (MB) restoration method with positivity constraint (PC) for 3D processing of data from 3D-SIM (or 3-beam interference SIM), in which the structured illumination pattern varies laterally and axially. The proposed 3D-MBPC method introduces positivity in the solution through the reconstruction of an auxiliary function using a conjugate-gradient method that minimizes the mean squared error between the data and the 3D imaging model. The 3D-MBPC method providesaxial super resolution, which is not the same as improved optical sectioning demonstrated with model-based approaches based on the 2D-SIM (or 2-beam interference SIM) imaging model, for either 2D or 3D processing of a single plane from a 3D-SIM dataset. Results obtained with our 3D-MBPC method show improved 3D resolution over what is achieved by the standard generalized Wiener filter method, the first known method that performs 3D processing of 3D-SIM data. Noisy simulation results quantify the achieved 3D resolution, which is shown to match theoretical predictions. Experimental verification of the 3D-MBPC method with biological data demonstrates successful application to data volumes of different sizes.

    

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3. Roadmap on Label‐Free Super‐Resolution Imaging

   https://doi.org/10.1002/lpor.202200029  

   Astratov, Vasily N. ; Sahel, Yair Ben ; Eldar, Yonina C. ; Huang, Luzhe ; Ozcan, Aydogan ; Zheludev, Nikolay ; Zhao, Junxiang ; Burns, Zachary ; Liu, Zhaowei ; Narimanov, Evgenii ; et al ( October 2023 , Laser & Photonics Reviews)

   Label‐free super‐resolution (LFSR) imaging relies on light‐scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super‐resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state‐of‐the‐art in this field, and to discuss the resolution boundaries and hurdles that need to be overcome to break the classical diffraction limit of the label‐free imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction‐limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super‐resolution capability that are based on understanding resolution as an information science problem, on using novel structured illumination, near‐field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere‐assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

    

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4. Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics

   https://doi.org/10.1038/s41467-021-23449-6  

   Lin, Ruizhe ; Kipreos, Edward T. ; Zhu, Jie ; Khang, Chang Hyun ; Kner, Peter ( May 2021 , Nature Communications)

   Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm throughCaenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.

    

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5. Signal, noise and resolution in linear and nonlinear structured‐illumination microscopy

   https://doi.org/10.1111/jmi.12753  

   INGERMAN, E. A. ; LONDON, R. A. ; HEINTZMANN, R. ; GUSTAFSSON, M. G. L. ( August 2018 , Journal of Microscopy)

   Structured‐illumination microscopy allows widefield fluorescence imaging with resolution beyond the classical diffraction limit. Its linear form extends resolution by a factor of two, and its nonlinear form by an in‐principle infinite factor, the effective resolution in practice being determined by noise. In this paper, we analyse the noise properties and achievable resolution of linear and nonlinear 1D and 2D patterned SIM from a frequency–space perspective. We develop an analytical theory for a general case of linear or nonlinear fluorescent imaging, and verify the analytical calculations with numerical simulation for a special case where nonlinearity is produced by photoswitching of fluorescent labels. We compare the performance of two alternative implementations, using either two‐dimensional (2D) illumination patterns or sequentially rotated one‐dimensional (ID) patterns. We show that 1D patterns are advantageous in the linear case, and that in the nonlinear case 2D patterns provide a slight signal‐to‐noise advantage under idealised conditions, but perform worse than 1D patterns in the presence of nonswitchable fluorescent background.

   Structured‐illumination microscopy (SIM) is a high‐resolution light microscopy technique that allows imaging of fluorescence at a resolution about twice the classical diffraction limit. There are various ways that the illumination can be structured, but it is not obvious how the choice of illumination pattern affects the final image quality, especially in view of the noise. We present a detailed performance analysis considering two illumination techniques: sequential illumination with line‐gratings that are shifted and rotated during image acquisition and two‐dimensional (2D) illumination structures requiring only shift operations. Our analysis is based on analytical theory, supported by simulations of images considering noise. We also extend our analysis to a nonlinear variant of SIM, with which enhanced resolution can be achieved, limited only by noise. This includes nonlinear SIM based on the light‐induced switching of the fluorescent molecules between a bright and a dark state. We find sequential illumination with line‐gratings to be advantageous in ordinary (linear) SIM, whereas 2D patterns provides a slight signal‐to‐noise advantage under idealised conditions in nonlinear SIM if there is no nonswitching background.

    

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