利用多色受激发射损耗（STED）显微镜获取呼​​吸道合胞体病毒颗粒和感染细胞的高分辨率图像

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

doi:10.21769/BioProtoc.2543

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

利用多色受激发射损耗（STED）显微镜获取呼吸道合胞体病毒颗粒和感染细胞的高分辨率图像

Masfique Mehedi ^* email

MS Margery Smelkinson ^*

JK Juraj Kabat

Sundar Ganesan

PC Peter L. Collins

UB Ursula J. Buchholz

(*contributed equally to this work) 发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2543 浏览次数: 9220

评审: Angela CoronaKristin ShinglerAnonymous reviewer(s)

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Cover of PLOS Pathogens, featuring study using the protocol.

Dec 2016

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Abstract

Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED increases resolution compared to confocal microscopy, which can be further improved by image processing using deconvolution software.

Keywords: RSV (RSV)

A549 (A549)

STED microscopy (STED显微镜技术)

Filopodia (丝状伪足)

Cell-to-cell spread (细胞间传播)

Immunofluorescence (免疫荧光)

Confocal microscopy (共聚焦显微镜技术)

Background

RSV forms pleomorphic virus particles, with a predominance of long filaments about 100 nm in diameter and up to about 10 µm in length (Bachi and Howe, 1973; Mehedi et al., 2016). High-resolution light microscopy techniques are key to visualizing the interactions between RSV infected cells and virus particles. In a recent study, we used super-resolution fluorescence microscopy to study RSV cell-to-cell spread in human lung epithelial A549 cells.
STED microscopy is one of the super-resolution microscopy techniques that have been developed to circumvent the limitations imposed by the ~200 nm diffraction barrier of light (Hell and Wichmann, 1994; Westphal et al., 2008). STED is based on confocal fluorescence microscopy and employs a pair of lasers, namely a pulsed excitation source and a photon depletion source. The excitation pulse is focused on the sample and excites the fluorescent dye therein. The excitation laser is superimposed with a doughnut-shaped STED depletion laser that quenches excited dye molecules except for the doughnut hole at the very center of the excitation focus, so that emission occurs only from the narrow center. Narrowing the excitation focal point in this way allows for images to be taken at resolutions far below the diffraction limit, e.g., typically 30-80 nm. While STED imaging relies on efficient dye depletion, image resolution and intensity are limited by photobleaching inflicted upon the dye. To address these two contrasting, yet key issues that arise with STED imaging, optimal sample preparation, most notably dye selection and signal intensity optimization, are crucial. STED enabled us to state conclusively that RSV was attached to filopodia rather than merely in the vicinity, and to precisely enumerate viral particles. Here, we describe how samples were prepared for multicolor STED imaging including dye selection, fixation procedure, imaging parameters, and deconvolution. We show how STED and STED deconvolution can improve lateral resolution both qualitatively and quantitatively.

Materials and Reagents

Aerosol resistant pipette tips
20 µl (Thermo Fisher Scientific, catalog number: 21-402-551 )
200 µl pipette tips (Thermo Fisher Scientific, catalog number: 02-682-255 )
1,000 µl pipette tips pipette tips (Thermo Fisher Scientific, catalog number: 21-402-582 )
T225 cm² flask with canted neck (Corning, Costar^®, catalog number: 3001 )
Microscope slides (super clean) (Scientific Device Laboratory, catalog number: 022 )
Sterile 12 mm circle untreated cover glasses; thickness 0.13-0.17 mm (Carolina Biological Supply, catalog number: 633029 )
50 ml conical tube
24-well cell culture plate (Corning, Costar^®, catalog number: 3524 )
The cell line of interest (human respiratory epithelial A549 cells [ATCC, catalog number: CCL-185 ])
Recombinant wild type RSV (A2 strain, GenBank KT992094) (virus stock with known virus titer, see Notes)
TryLE Select cell dissociation reagent, stored at room temperature (Thermo Fisher Scientific, Gibco^TM, catalog number: 12563 )
Bovine serum albumin (BSA) standard (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23210 )
Anti-RSV F protein mouse monoclonal antibody (mAb) (Abcam, catalog number: ab43812 )
Anti-beta-tubulin (9F3) rabbit mAb (Cell Signaling Technology, catalog number: 2128 )
Goat anti-mouse Alexa Fluor 488 (AF488) (Thermo Fisher Scientific, catalog number: A11029 )
Goat anti-rabbit IgG-Atto 647N (Sigma-Aldrich, catalog number: 40839 )
Rhodamine phalloidin (CYTOSKELETON, catalog number: PHDR1 )
ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36930 )
Ultrapure methanol free formaldehyde prepared from paraformaldehyde (PFA) 16% solution, EM Grade (Polysciences, catalog number: 18814 )
F-12 medium without additives, which is sold commercially as Ham’s F-12 nutrient mix (Thermo Fisher Scientific, Gibco^TM, catalog number: 11765054 )
Fetal bovine serum (FBS) (GE Healthcare, HyClone^TM, catalog number: SH30071.03 )
L-Glutamine 200 mM (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030081 )
Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190 )
Triton X-100 (BioUltra, ~10% in H₂O, Sigma-Aldrich, catalog number: 93443 )
Trypan blue 0.4% solution (Lonza, catalog number: 17-942E )
F-12 complete medium (see Recipes)
4% PFA (see Recipes)
0.05% Triton X-100 (see Recipes)
3% BSA (see Recipes)

Equipment

Pipettes (Mettler-Toledo, RAININ, model: Pipet-Lite XLS )
Humidified CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM Steri-Cult^TM )
Centrifuge (Beckman Coulter, model: Allegra 25R )
Leica TCS SP8 STED 3X system (Leica Microsystems, model: Leica TCS SP8 STED 3X ) equipped with:
1. A white light excitation laser
2. 592 nm, 600 nm, 775 nm depletion lasers
3. HC PL APO 100x/1.40 oil STED white objective
4. Gated HyD hybrid detectors
Hemocytometer (Marienfeld-Superior, catalog number: 0680030 )
Dumont NOC tweezer (Electron Microscopy Sciences, catalog number: 0103-NOC-PO-1 )

Software

Images were acquired using LAS X software (version 3.1.1.15751) (Leica Microsystems)
Images were deconvolved using Huygens deconvolution software (Huygens Essentials version 16.10.1.p3, Scientific Volume Imaging BV, Hilversum, The Netherlands)
PRISM software version 7

Procedure

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Mehedi, M., Smelkinson, M., Kabat, J., Ganesan, S., Collins, P. L. and Buchholz, U. J. (2017). Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells. Bio-protocol 7(17): e2543. DOI: 10.21769/BioProtoc.2543.