1201009_Rozario,A_2022.pdf (3.38 MB)
3D Single Molecule Super-Resolution Microscopy of Whole Nuclear Lamina
journal contributionposted on 2022-06-17, 05:13 authored by Ashley RozarioAshley Rozario, A Morey, C Elliott, B Russ, Donna WhelanDonna Whelan, SJ Turner, TDM Bell
Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.
& nbsp;Support from the National Health and Medical Research Council through the Ideas program (GA65914) is gratefully acknowledged. DW is the recipient of an Australian Research Council Australian Discovery Early Career Research Award (DE200100584) funded by the Australian Government.
JournalFrontiers in Chemistry
PublisherFrontiers Media S.A.
Rights Statement© 2022 Rozario, Morey, Elliott, Russ, Whelan, Turner and Bell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms
Science & TechnologyPhysical SciencesChemistry, MultidisciplinaryChemistryastigmatismmultiplane imagingT cellnuclear envelopeconvex hull3-DIMENSIONAL SUPERRESOLUTIONSUBDIFFRACTION-RESOLUTIONEXPANSION MICROSCOPYDIFFRACTION-LIMITIMAGING REVEALSCELLENVELOPEORGANIZATIONPROTEINSTARGETPhysical Sciences not elsewhere classified