Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells

Mehedi, Masfique; Smelkinson, Margery; Kabát, Juraj; Ganesan, Sundar; Collins, Peter L.; Buchholz, Ursula J.

doi:10.21769/bioprotoc.2543

Cited by 15 publications

(16 citation statements)

References 6 publications

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“…Previously, it has also shown that the cytoskeleton protein actin is involved in RSV endocytosis, replication, gene expression, and morphogenesis (Figure 2) [36][37][38][39]. It has recently been shown that ARP2/3 and virus-induced filopodia contribute to RSV cell-to-cell spread (Figures 2 and 3) [40][41][42].…”

Section: Introductionmentioning

confidence: 91%

Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection

et al. 2021

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Respiratory syncytial virus (RSV) is the leading viral agent causing bronchiolitis and pneumonia in children under five years old worldwide. The RSV infection cycle starts with macropinocytosis-based entry into the host airway epithelial cell membrane, followed by virus transcription, replication, assembly, budding, and spread. It is not surprising that the host actin cytoskeleton contributes to different stages of the RSV replication cycle. RSV modulates actin-related protein 2/3 (ARP2/3) complex-driven actin polymerization for a robust filopodia induction on the infected lung epithelial A549 cells, which contributes to the virus’s budding, and cell-to-cell spread. Thus, a comprehensive understanding of RSV-induced cytoskeletal modulation and its role in lung pathobiology may identify novel intervention strategies. This review will focus on the role of the ARP2/3 complex in RSV’s pathogenesis and possible therapeutic targets to the ARP2/3 complex for RSV.

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Section: Introductionmentioning

confidence: 91%

Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection

et al. 2021

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“…Thus, obtaining images with high resolution and contrast coupled with acceptable low cross-talk values is a rather challenging task. Different approaches have been developed in the past to overcome this limitation: the implementation of more than one STED laser, 10,25,39 the usage of long Stokes shift fluorophores, 26,40 or spectral unmixing. 29 The implementation of more than one STED laser leads to a complex optical system.…”

Section: ■ Discussionmentioning

confidence: 99%

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

et al. 2021

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Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

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“…This technique has been recently used in neuroscience to better visualize dendritic cells and understand their structure and function ( 24 ). STED has been applied to lung tissue by detecting filamentous human respiratory syncytial virus particles, providing a better understanding and mechanism of how the virus spreads cell to cell (e.g., microbiota activity) ( 25 ). STED microscopy can potentially aid in understanding the molecular mechanisms of receptors and their specific ligands in disease, such as pulmonary hypertension, fibrosis and malignancies ( 26 , 27 ).…”

Section: Discussionmentioning

confidence: 99%

From 2D to 3D: Promising Advances in Imaging Lung Structure

et al. 2020

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The delicate structure of murine lungs poses many challenges for acquiring high-quality images that truly represent the living lung. Here, we describe several optimized procedures for obtaining and imaging murine lung tissue. Compared to traditional paraffin cross-section and optimal cutting temperature (OCT), agarose-inflated vibratome sections (aka precision-cut lung slices), combines comparable structural preservation with experimental flexibility. In particular, we discuss an optimized procedure to precision-cut lung slices that can be used to visualize three-dimensional cell-cell interactions beyond the limitations of two-dimensional imaging. Super-resolution microscopy can then be used to reveal the fine structure of lung tissue's cellular bodies and processes that regular confocal cannot. Lastly, we evaluate the entire lung vasculature with clearing technology that allows imaging of the entire volume of the lung without sectioning. In this manuscript, we combine the above procedures to create a novel and evolutionary method to study cell behavior ex vivo , trace and reconstruct pulmonary vasculature, address fundamental questions relevant to a wide variety of vascular disorders, and perceive implications to better imaging clinical tissue.

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Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells

Cited by 15 publications

References 6 publications

Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection

Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

From 2D to 3D: Promising Advances in Imaging Lung Structure

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