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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   

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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

  1. 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 )
  2. T225 cm2 flask with canted neck (Corning, Costar®, catalog number: 3001 )
  3. Microscope slides (super clean) (Scientific Device Laboratory, catalog number: 022 )
  4. Sterile 12 mm circle untreated cover glasses; thickness 0.13-0.17 mm (Carolina Biological Supply, catalog number: 633029 )
  5. 50 ml conical tube
  6. 24-well cell culture plate (Corning, Costar®, catalog number: 3524 )
  7. The cell line of interest (human respiratory epithelial A549 cells [ATCC, catalog number: CCL-185 ])
  8. Recombinant wild type RSV (A2 strain, GenBank KT992094) (virus stock with known virus titer, see Notes)
  9. TryLE Select cell dissociation reagent, stored at room temperature (Thermo Fisher Scientific, GibcoTM, catalog number: 12563 )
  10. Bovine serum albumin (BSA) standard (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23210 )
  11. Anti-RSV F protein mouse monoclonal antibody (mAb) (Abcam, catalog number: ab43812 )
  12. Anti-beta-tubulin (9F3) rabbit mAb (Cell Signaling Technology, catalog number: 2128 )
  13. Goat anti-mouse Alexa Fluor 488 (AF488) (Thermo Fisher Scientific, catalog number: A11029 )
  14. Goat anti-rabbit IgG-Atto 647N (Sigma-Aldrich, catalog number: 40839 )
  15. Rhodamine phalloidin (CYTOSKELETON, catalog number: PHDR1 )
  16. ProLong Gold Antifade Mountant (Thermo Fisher Scientific, InvitrogenTM, catalog number: P36930 )
  17. Ultrapure methanol free formaldehyde prepared from paraformaldehyde (PFA) 16% solution, EM Grade (Polysciences, catalog number: 18814 )
  18. F-12 medium without additives, which is sold commercially as Ham’s F-12 nutrient mix (Thermo Fisher Scientific, GibcoTM, catalog number: 11765054 )
  19. Fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30071.03 )
  20. L-Glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  21. Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190 )
  22. Triton X-100 (BioUltra, ~10% in H2O, Sigma-Aldrich, catalog number: 93443 )
  23. Trypan blue 0.4% solution (Lonza, catalog number: 17-942E )
  24. F-12 complete medium (see Recipes)
  25. 4% PFA (see Recipes)
  26. 0.05% Triton X-100 (see Recipes)
  27. 3% BSA (see Recipes)

Equipment

  1. Pipettes (Mettler-Toledo, RAININ, model: Pipet-Lite XLS )
  2. Humidified CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CultTM )
  3. Centrifuge (Beckman Coulter, model: Allegra 25R )
  4. 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
  5. Hemocytometer (Marienfeld-Superior, catalog number: 0680030 )
  6. Dumont NOC tweezer (Electron Microscopy Sciences, catalog number: 0103-NOC-PO-1 )

Software

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

Procedure

  1. Virus infection
    1. Maintain A549 cells in T225 flasks, following the ATCC’s culturing recommendations, with the following modifications:
      1. Use F-12 complete medium to maintain the line (see Recipes); use TrypLE Select cell dissociation reagent; use FBS to inactivate the TrypLE Select.
      2. To seed A549 cells onto coverslips, remove the F12 complete cell culture medium from a T225 flask containing a monolayer of about 80% confluent A459 cells. Gently rinse the monolayer with 10 ml pre-warmed (37 °C) 1x DPBS, and remove the DPBS.
      3. To dissociate cells, add 5 ml TryLE Select cell dissociation reagent, and incubate for 5 min at 37 °C in a standard humidified CO2 incubator. Inactivate TrypLE Select by adding 10 ml of cold FBS. Detach and dissociate A549 cells by gentle pipetting, and transfer the single-cell suspension to a 50 ml conical tube.
      4. Pellet the cells by centrifugation at 300 x g for 5 min at 4 °C in a tabletop centrifuge. Resuspend the cell pellet in 10 ml of pre-warmed (37 °C) F-12 complete medium. Dilute 10 µl of the resuspended A549 cells 1:10 in DPBS with 0.04% trypan blue (80 µl DPBS,10 µl trypan blue [0.4%],10 µl aliquot of A549 cells, resuspended in complete F-12 medium) and determine the concentration of the resuspended cells per ml using a hemocytometer as recommended by the manufacturer.
      5. Dilute the resuspended A549 cells from the 50 ml conical tube with F-12 complete medium to 3 x 104 cells per ml, and seed cells by adding 1 ml of F-12 complete medium with 3 x 104 cells to each well of a 24-well plate containing a coverslip.
      6. Incubate the cells overnight in a cell culture incubator at 37 °C, 5% CO2.
    2. To infect A549 cells, replace the medium with 150 µl F-12 medium without additives, containing sucrose-purified RSV (Collins et al., 1995 and Le Nouen et al., 2009; see Notes) at a multiplicity of infection (MOI) of 1 plaque forming unit (PFU) per cell (see Notes).
    3. Incubate A549 cells with virus inoculum for 1 h at 37 °C in a CO2 incubator.
    4. Remove virus inoculum and wash infected cells 2 x with 1 ml F-12 medium without additives.
    5. Incubate infected cells in 1 ml of F-12 medium with 2% FBS and 1% L-glutamine for 24 h at 37 °C in a CO2 incubator.

  2. Slide preparation
    1. Remove cell culture medium and wash monolayers 3 x with 1 ml DPBS.
    2. To fix infected cells, incubate cells with 1 ml of a freshly prepared 1:4 dilution of PFA (4% final concentration) in DPBS for 30 min at room temperature.
    3. Remove PFA solution and wash cells 3 x each with 1 ml DPBS.
    4. To permeabilize fixed cells, incubate with 1 ml 0.05% Triton X-100 in DPBS for 10 min at room temperature.
    5. Remove Triton X-100 solution and wash cells 3 x each with 1 ml DPBS.
    6. To block unspecific protein binding, incubate coverslip with 1 ml 3% BSA in PBS for 3 h at 4 °C.
    7. Wash cells 1 x with 1 ml DPBS.
    8. Incubate cells overnight at 4 °C with primary antibody mix: mouse anti-RSV F mAb (1:500) and rabbit anti-tubulin mAb (1:100) in PBS with 0.1% BSA.
    9. Wash cells 3 x each with 1 ml DPBS.
    10. Incubate cells with secondary antibody mix: goat anti-mouse AF488 (1:200) and goat anti-rabbit Atto 647N (1:100) in DPBS with 0.1% BSA for 3 h at 4 °C in the dark.
    11. Wash cells 3 x each with 1 ml DPBS.
    12. For actin cytoskeleton staining, incubate cells with rhodamine phalloidin (1:500) in DPBS for 30 min at 4 °C in the dark.
    13. Wash cells 2 x each with 1 ml DPBS.
    14. Wash cells 2 x each with 1 ml deionized H2O.
    15. To mount the coverslip on a glass slide, first place 10 µl of ProLong Gold Antifade mounting medium onto a glass slide. Pick up the coverslip with Dumont #NO forceps and lower the cover slip cell-side down onto the mounting medium, taking care to avoid trapping air bubbles.
    16. Dry slide overnight in the dark.

  3. Imaging procedure
    1. In Figure 1, all images were collected in a single focal plane with a 30 nm pixel size using a bidirectional scan speed of 600 Hz.


      Figure 1. STED imaging of RSV viral particles along filopodia. A549 cells were infected with RSV (MOI = 1 PFU/cell) for 24 h. Cells were fixed, permeabilized, and immunostained with antibodies for RSV F (green); the cellular tubulin network was visualized by staining for beta tubulin (cyan). These cells were further stained with rhodamine phalloidin to detect F-actin (red). All images were collected in a single focal plane as described in the imaging procedure. For confocal image acquisition (left panel), excitation wavelengths for the detection of RSV F (AF488), F-actin (rhodamine phalloidin), and tubulin (Atto 647N) were 488 nm, 561 nm, and 647 nm, respectively. The subsequent STED acquisition (middle panel) was done in the following order: first, the Atto 647N conjugate used for tubulin immunostaining was excited with 647 nm and depleted with 775 nm; then, to visualize F-actin, rhodamine phalloidin was excited with 561 nm and depleted with 660 nm; and finally, the AF488 conjugate used for RSV F specific immunostaining was excited with 488 nm and depleted with 592 nm. Filopodia (indicated by arrows on the bottom left image) appear to shuttle RSV particles towards a neighboring cell (marked with a * in the bottom left image). An increase in resolution of all channels is apparent with STED imaging, which can be further improved by deconvolution using Huygens software (STED decon, right panel). Scale bar = 3 µm, inset scale bar = 1 µm.

    2. Gated HyD detectors were used to collect an emission bandwidth of approximately 40 nm. All fluorophores were excited with a pulsed white light laser tuned to the appropriate wavelength.
    3. For confocal channel acquisition, AF488 was excited with 488 nm; rhodamine phalloidin was excited with 561 nm; and Atto 647N was excited with 647 nm. All HyD detector gating was set to 0.3-6 nsec. High intensity signals enabled the usage of low laser power settings, and single color controls were used to confirm the absence of background fluorescence. Of note, the laser power for confocal acquisition was set 3-5 fold lower than for STED acquisition. Photobleaching during confocal acquisition was negligible.
    4. Photon depletion occurs when some overlap exists between the STED laser wavelength and the emission spectrum of the fluorophore. Thus, for STED channel acquisition (subsequent to confocal acquisition), AF488 was excited with 488 nm and photon depleted with 592 nm with 1.2-6 nsec HyD gating; rhodamine phalloidin was excited with 561 nm and depleted with 660 nm with 1-6 nsec HyD gating; and Atto 647N was excited with 647 nm and depleted with 775 nm with 0.6-6 nsec HyD gating.
    5. Caution needs to be taken to limit exposure of fluorophores to a depletion laser if they can absorb energy at that specific wavelength (i.e., fluorophores that have excitation spectrums encompassing the wavelengths of the depletion lasers, 592 nm, 660 nm, or 775 nm). This is because the depletion laser output at the imaging plane is ~500-1,000 times greater than the excitation source, which will rapidly result in photobleaching. For example, Atto 647N will be photobleached by the 660 nm depletion laser, Alexa 594 will be photobleached by the 592 nm depletion laser, etc. Therefore, to avoid photobleaching, the collection order of the STED channels is of utmost importance, with longer wavelength fluorophores collected first and the shorter wavelengths collected last. In this experiment, the collection order of the STED channels was Atto 647N, followed by rhodamine phalloidin, and then AF488.
    6. Using single color controls, we confirmed that only the intended fluorophore was excited per each channel, and that neither cellular autofluorescence nor non-specific binding of the primary or secondary antibodies were detected.
    7. While confocal images were collected with a pinhole set to 1 Airy Unit (AU), this was reduced to 0.7 AU for STED imaging to reduce optical sectioning and increase the signal-to-noise ratio.
    8. Additionally, due to the strong depletion power, excitation powers were increased approximately 3-5 fold compared to confocal to compensate for signal loss. A frame accumulation of 2 was also used to further amplify the signal.

  4. Image processing
    1. STED images were deconvolved using Huygens deconvolution software (Huygens Essentials v.17.040.p5, SVI BV, The Netherlands) to reverse the optical distortion created during image acquisition (Figure 2). Within this software package, we used the Deconvolution Wizard with automatic background subtraction and microscopic parameters recognition with a continued maximum likelihood estimate (CMLE) iterative algorithm. Processing parameters included microscopic and deconvolution parameters, which are important for proper point spread function (PSF) calculation necessary for successful deconvolution. Microscopic parameters were verified and corrected if necessary to avoid processing artifacts. The deconvolution parameters were adjusted in this Huygens deconvolution package as described below. This Huygens Essentials package was the only STED deconvolution package available at that time. However, there now are a number of software packages or free plug-ins available for classical confocal, multiphoton, or wide-field deconvolution applications.


      Figure 2. STED and deconvolution improve lateral resolution. To quantify the improvement in image resolution shown in Figure 1, we took the Gaussian profiles (bell curves) of signal intensity for a number of representative narrow structures and measured the width of the curve at the intensity level that is half of the maximum, which provided ‘full-width at half-maximum’ (FWHM) values. Smaller values of FWHM indicate improved resolution. This was done for five structures from each channel (green: virus filaments (RSV F), red: actin filaments, and cyan: tubulin filaments). The same structures were measured in confocal, STED, and STED deconvolved (STED decon) images. For each stain, STED significantly improved resolution compared to confocal microscopy, and resolution was further improved by deconvolution. Error bars represent standard deviation.

    2. STED resolution was improved in Huygens Essentials by using settings for background subtraction, for the number of iterations, and for the desired signal-to-noise ratios that were determined empirically after several rounds of iterations. For RSV F, these values were 0.0316 for background subtraction, 54 for the number of iterations, and 18 for the signal-to-noise ratio. For F-actin, these values were 0.0695, 46, and 19, respectively. For tubulin, these values were 0.0835, 49, and 20, respectively. Deconvolved images were thoroughly compared with the original raw image to avoid artifacts such as striping, ringing, or discontinuous staining.
    3. Additional deconvolution parameters were set in Huygens Essentials to account for the expected amount by which the fluorescence is suppressed by the STED beam (STED saturation factor) and the expected fraction of fluorescent molecules that is photoresistant to the depletion beam (STED immunity fraction). The STED saturation factor is the absolute intensity of the STED laser divided by the saturation intensity. For the maximum intensity STED laser, the saturation factor is 30, and it is scaled based on the STED laser intensity. The STED immunity fraction is the fraction of the fluorophores that has not been depleted by the STED laser. It is described as an additional confocal PSF component that is added to the pure STED PSF and is estimated in percentage of the maximum saturation. It has only a minor influence on the quality of deconvolved images, and typical values are around 10%. For RSV F, these values were 30 for the STED saturation factor, and 14 for the STED immunity fraction. For F-actin, these values were 27 and 10, respectively. For tubulin, these values were 5 and 11, respectively.

Notes

  1. To avoid disturbing the cell monolayer while pipetting, tilt the plate slightly (at an angle less than 45°) and direct the pipette tip towards the side wall of each well during dispensing.
  2. Pipette slowly to preserve fragile cellular structures and viral filaments.
  3. To prevent possible interfering effects on cell biology, do not include antibiotic and anti-fungal agents when culturing A549 cells
  4. It is not essential that sucrose purified recombinant RSV be used for these studies.
  5. Prior to these studies, the titer of the RSV stock should be determined by immunoplaque assay on 24-well plates of Vero 76 cells (ATCC CRL-1587). In short, prepare serial tenfold dilutions of the RSV stock of interest in cell culture medium. Discard the growth medium from 24-well plates of subconfluent Vero cell monolayers, and transfer 100 µl per well of the serial RSV dilutions to duplicate wells of 24-well plates. To allow for virus adsorption, incubate for 2 h in a cell culture incubator; gently rocking the plates every 20 min to prevent the monolayers from drying out. After adsorption, overlay the monolayers with 1 ml of 0.8% methyl cellulose overlay (prepared using cell culture medium) per well, and incubate the cultures for 5-6 days. Then discard the methyl cellulose overlay by inverting the plates, and fix with ice-cold 80% methanol. Visualize RSV plaques by immunostaining using an RSV specific primary antibody preparation (for example a commercially available mouse monoclonal antibody to RSV), followed by a species-specific secondary antibody (for example, a peroxidase-conjugated goat anti-mouse IgG(H+L), KPL #074-18064), and a detection system of choice (for example, CN Peroxidase substrate, KPL #50-73-02, and peroxidase substrate solution B, KPL #50-65-02). Select wells of a dilution that yielded well-separated plaques, count virus plaques per well, and calculate the titer of the virus stock (plaque forming units per ml) by multiplying the average number of plaques by the dilution factor.

Data analysis

Images were deconvolved and FWHM measurements were made using Huygens deconvolution software. Standard deviation was calculated using PRISM software version 7. STED microscopic observation of filopodia-driven RSV cell-to-cell spread has previously been described in detail in (Mehedi et al., 2016). Here, we include an additional channel to visualize the tubulin network in the RSV infected cells.

Recipes

  1. F-12 complete medium
    500 ml F-12 nutrient mix
    10% FBS
    1% L-glutamine
  2. 4% PFA
    30 ml 1x DPBS
    10 ml 16% PFA
  3. 0.05% Triton X-100
    50 ml 1x DPBS
    250 µl Triton X-100
  4. 3% BSA
    50 ml 1x DPBS
    1,500 µl BSA (1 mg/ml)

Acknowledgments

This study was supported by the Intramural Research Program of NIAID, NIH. All authors have declared that no competing interest exists. This protocol was adopted from previously published work (Mehedi et al., 2016).

References

  1. Bachi, T. and Howe, C. (1973). Morphogenesis and ultrastructure of respiratory syncytial virus. J Virol 12(5): 1173-1180.
  2. Collins, P. L., Hill, M. G., Camargo, E., Grosfeld, H., Chanock, R. M. and Murphy, B. R. (1995). Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5’ proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci U S A 92(25): 11563-11567.
  3. Hell, S. W. and Wichmann, J. (1994). Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11): 780-782.
  4. Le Nouen, C., Munir, S., Losq, S., Winter, C. C., McCarty, T., Stephany, D. A., Holmes, K. L., Bukreyev, A., Rabin, R. L., Collins, P. L. and Buchholz, U. J. (2009). Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3. Virology 385(1): 169-182.
  5. Mehedi, M., McCarty, T., Martin, S. E., Le Nouen, C., Buehler, E., Chen, Y. C., Smelkinson, M., Ganesan, S., Fischer, E. R., Brock, L. G., Liang, B., Munir, S., Collins, P. L. and Buchholz, U. J. (2016). Actin-related protein 2 (ARP2) and virus-induced filopodia facilitate human respiratory syncytial virus spread. PLoS Pathog 12(12): e1006062.
  6. Westphal, V., Rizzoli, S. O., Lauterbach, M. A., Kamin, D., Jahn, R. and Hell, S. W. (2008). Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320(5873): 246-249.

简介

人肺上皮A549细胞中的呼吸道合胞病毒(RSV)感染诱导丝状伪足,由F-肌动蛋白组成的细胞突起,延伸至相邻的未感染细胞(Mehedi等,2016)。 通过受激发射耗尽(STED)显微镜的高分辨率成像显示沿着这些丝状伪足的丝状RSV颗粒,表明丝状伪足有助于RSV细胞对细胞的扩散(Mehedi等,2016)。 在本协议中,我们描述如何修复,渗透,免疫染色和挂载RSV感染的A549细胞进行STED成像。 我们显示与共聚焦显微镜相比,STED增加了分辨率,可以通过使用去卷积软件的图像处理进一步改进。
【背景】RSV形成多形性病毒颗粒,其长度大约为直径约100nm,长度大约为10μm(Bachi和Howe,1973; Mehedi等,2016)。高分辨率光学显微技术是可视化RSV感染细胞和病毒颗粒之间相互作用的关键。在最近的一项研究中,我们使用超分辨率荧光显微镜来研究人肺上皮A549细胞中的RSV细胞对细胞的扩散。
  STED显微镜是超分辨率显微镜技术之一,已被开发以规避约200nm衍射屏障的光限制(Hell和Wichmann,1994; Westphal等人,2008)。 STED基于共焦荧光显微镜,并采用一对激光器,即脉冲激发光源和光子耗尽源。激发脉冲聚焦在样品上并激发荧光染料。激发激光器与环形STED耗尽激光器叠加,激发染料分子除了在激发焦点的中心处的环形孔外,使得发射仅发生在狭窄的中心。以这种方式缩小激发焦点允许以远低于衍射极限的分辨率拍摄图像,例如通常为30-80nm。虽然STED成像依赖于有效的染料消耗,但是图像分辨率和强度受染料染色后的光漂白的限制。为了解决STED成像产生的两个对比但关键问题,最佳样品制备,最着名的是染料选择和信号强度优化,至关重要。 STED使我们能够断言,RSV是附加于丝状伪足而不仅仅是在附近,并精确地列举病毒颗粒。在这里,我们描述了如何为多色STED成像准备样品,包括染料选择,固色程序,成像参数和去卷积。我们展示了STED和STED去卷积如何能够在定性和定量上提高横向分辨率。

关键字:RSV, A549, STED显微镜技术, 丝状伪足, 细胞间传播, 免疫荧光,  共聚焦显微镜技术

材料和试剂

  1. 气溶胶移液管吸头
    20μl(Thermo Fisher Scientific,目录号:21-402-551)
    200μl移液器吸头(Thermo Fisher Scientific,目录号:02-682-255)
    1000μl移液器吸头吸头(Thermo Fisher Scientific,目录号:21-402-582)
  2. 具有斜颈的T225厘米烧瓶(Corning,Costar ®,目录号:3001)
  3. 显微镜幻灯片(超级清洁)(Scientific Device Laboratory,目录号:022)
  4. 无菌12毫米圆未经处理的护目镜;厚度0.13-0.17mm(Carolina Biological Supply,目录号:633029)
  5. 50ml圆锥管
  6. 24孔细胞培养板(Corning,Costar ®,目录号:3524)
  7. 感兴趣的细胞系(人呼吸上皮A549细胞[ATCC,目录号:CCL-185])
  8. 重组野生型RSV(A2株,GenBank KT992094)(具有已知病毒滴度的病毒库,参见注释)
  9. TryLE选择在室温下储存的细胞解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:12563)
  10. 牛血清白蛋白(BSA)标准品(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23210)
  11. 抗RSV F蛋白小鼠单克隆抗体(mAb)(Abcam,目录号:ab43812)
  12. 抗β-微管蛋白(9F3)兔mAb(Cell Signaling Technology,目录号:2128)
  13. 山羊抗小鼠Alexa Fluor 488(AF488)(Thermo Fisher Scientific,目录号:A11029)
  14. 山羊抗兔IgG-Atto 647N(Sigma-Aldrich,目录号:40839)
  15. 罗丹明鬼笔环肽(CYTOSKELETON,目录号:PHDR1)
  16. ProLong Gold Antifade Mountant(Thermo Fisher Scientific,Invitrogen TM ,目录号:P36930)
  17. 由多聚甲醛(PFA)制备的超纯甲醇游离甲醛16%溶液,EM级(Polysciences,目录号:18814)
  18. F-12无添加剂的介质,以Ham's F-12营养组合(Thermo Fisher Scientific,Gibco TM,目录号:11765054)商业销售
  19. 胎牛血清(FBS)(GE Healthcare,HyClone TM,目录号:SH30071.03)
  20. L-谷氨酰胺200mM(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  21. Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190)
  22. Triton X-100(BioUltra,在H 2 O中约10%,Sigma-Aldrich,目录号:93443)
  23. 台盼蓝0.4%溶液(Lonza,目录号:17-942E)
  24. F-12完整培养基(见食谱)
  25. 4%PFA(见配方)
  26. 0.05%Triton X-100(参见食谱)
  27. 3%BSA(参见食谱)

设备

  1. 移液器(Mettler-Toledo,RAININ,型号:Pipet-Lite XLS)
  2. 加湿的CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific&< sup>,型号:Forma< sup>< Steri-Cult<
  3. 离心机(Beckman Coulter,型号:Allegra 25R)
  4. Leica TCS SP8 STED 3X系统(Leica Microsystems,型号:Leica TCS SP8 STED 3X)配备:
    1. 白光激发激光器
    2. 592 nm,600 nm,775 nm耗尽激光器
    3. HC PL APO 100x / 1.40油STED白色物镜
    4. 门式HyD混合式探测器
  5. 血细胞计数器(Marienfeld-Superior,目录号:0680030)
  6. 杜蒙NOC镊子(电子显微镜科学,目录号:0103-NOC-PO-1)

软件

  1. 使用LAS X软件(版本3.1.1.15751)(Leica Microsystems)
    获取图像
  2. 使用惠更斯反卷积软件(Huygens Essentials版本16.10.1.p3,Scientific Volume Imaging BV,Hilversum,荷兰)对图像进行去卷积
  3. PRISM软件版本7

程序

  1. 病毒感染
    1. 按照ATCC的培养建议,维持T225烧瓶中的A549细胞,并进行以下修改:
      1. 使用F-12完整培养基维持生产线(参见食谱);使用TrypLE选择细胞分离试剂;使用FBS禁用TrypLE选择。
      2. 为了将A549细胞种在盖玻片上,从含有约80%汇合的A459细胞的单层的T225烧瓶中除去F12完全细胞培养基。用10ml预热(37℃)1x DPBS轻轻冲洗单层,并除去DPBS。
      3. 为了解离细胞,加入5ml TryLE Select细胞解离试剂,并在37℃下在标准加湿的CO 2培养箱中孵育5分钟。通过加入10ml冷FBS灭活TrypLE选择。通过轻轻移液分离和解离A549细胞,并将单细胞悬浮液转移到50ml锥形管中。
      4. 通过在桌面离心机中在4℃下以300×g离心5分钟来将细胞造粒。将细胞沉淀重悬在10ml预热(37℃)F-12完全培养基中。稀释10μl重悬的A549细胞1:10在含有0.04%台盼蓝的DPBS中(80μlDPBS,10μl台盼蓝[0.4%],10μl等份的A549细胞,重悬于完全F-12培养基中),并确定使用制造商推荐的血细胞计数器,每ml重悬细胞的浓度。
      5. 用50ml F-12完全培养基至3×10 4个/ ml细胞/ ml的50ml锥形管稀释重悬的A549细胞,通过加入1ml具有3×10 4 细胞到含有盖玻片的24孔板的每个孔中
      6. 在37℃,5%CO 2的细胞培养箱中孵育细胞一夜。
    2. 为了感染A549细胞,用不含添加剂的150μlF-12培养基代替培养基,其中含有蔗糖纯化的RSV(Collins等人,1995和Le Nouen等人)。 ,2009;见注释),每个细胞1个噬菌斑形成单位(PFU)的感染复数(MOI)(见注释)。
    3. 在37℃的CO 2培养箱中,用病毒接种物孵育A549细胞1小时。
    4. 去除病毒接种物,并用1ml F-12培养基洗涤感染细胞2×,无添加剂。
    5. 将感染的细胞在含有2%FBS和1%L-谷氨酰胺的1ml F-12培养基中在37℃下在CO 2培养箱中孵育24小时。

  2. 幻灯片准备
    1. 取出细胞培养基,用1ml DPBS洗涤3次
    2. 为了固定受感染的细胞,在室温下,用1ml新鲜制备的1:4稀释度的PFA(4%终浓度)在DPBS中孵育细胞30分钟。
    3. 取出PFA溶液,每孔用1ml DPBS洗涤3次
    4. 为了透化固定的细胞,在室温下用DPBS中的1ml 0.05%Triton X-100孵育10分钟。
    5. 取出Triton X-100溶液,每孔用1ml DPBS洗涤3次
    6. 为了阻止非特异性蛋白质结合,用PBS中的1ml 3%BSA在4℃孵育盖玻片3小时。
    7. 用1ml DPBS洗涤细胞1×。
    8. 在含有0.1%BSA的PBS中,在4℃孵育细胞过夜,使用一抗抗体混合物:小鼠抗RSV F mAb(1:500)和兔抗微管蛋白mAb(1:100)。
    9. 洗涤细胞3次,每次1ml DPBS。
    10. 在含有0.1%BSA的DPBS中,在4℃下在黑暗中孵育具有二抗混合物的细胞:山羊抗小鼠AF488(1:200)和山羊抗兔Atto 647N(1:100)3小时。
    11. 洗涤细胞3次,每次1ml DPBS。
    12. 对于肌动蛋白细胞骨架染色,在DPBS中将罗丹明鬼笔环肽(1:500)细胞在4℃黑暗中孵育30分钟。
    13. 洗涤细胞2 x每个1 ml DPBS。
    14. 用1ml去离子H 2 O 2洗涤2×2个细胞。
    15. 要将盖玻片安装在玻璃片上,首先将10μlProLong Gold Antifade安装介质放在玻璃片上。用Dumont #NO镊子取下盖玻片,将盖板单元向下放到安装介质上,注意避免吸入气泡。
    16. 在黑暗中干燥过夜。

  3. 成像程序
    1. 在图1中,使用600Hz的双向扫描速度将所有图像收集在具有30nm像素尺寸的单焦点平面中。


      图1. RSV病毒颗粒沿丝状伪足的STED成像 A549细胞用RSV(MOI = 1PFU /细胞)感染24小时。细胞用RSV F(绿色)的抗体固定,透化和免疫染色;细胞微管蛋白网络通过染色β微管蛋白(青色)而显现。用罗丹明鬼笔环肽进一步染色这些细胞以检测F-肌动蛋白(红色)。如成像过程中所述,将所有图像收集在单个焦平面上。对于共焦图像采集(左图),用于检测RSV F(AF488),F-肌动蛋白(罗丹明鬼笔环肽)和微管蛋白(Atto 647N)的激发波长分别为488nm,561nm和647nm。随后的STED采集(中图)按以下顺序进行:首先,用于微管蛋白免疫染色的Atto 647N缀合物用647nm激发并耗尽775nm;然后,为了可视化F-肌动蛋白,罗丹明鬼笔环肽用561nm激发并耗尽660nm;最后,用于RSV F特异性免疫染色的AF488缀合物用488nm激发并耗尽592nm。 Filopodia(左下角的箭头指示)似乎将RSV颗粒穿过邻近的小区(在左下角的图像中用*标记)。所有通道的分辨率的增加是显而易见的STED成像,可以通过使用惠更斯软件(STED decon,右图)的去卷积进一步改善。比例尺= 3μm,插图比例尺= 1μm。

    2. 门控HyD探测器用于收集大约40nm的发射带宽。所有的荧光团都用调谐到适当波长的脉冲白光激光器激发。
    3. 对于共焦信道采集,AF488激发488 nm;罗丹明鬼笔环肽用561nm激发;并用647nm激发Atto 647N。所有HyD探测器门控设置为0.3-6 ns。高强度信号使得能够使用低激光功率设置,并且使用单色控制来确认不存在背景荧光。值得注意的是,用于共聚焦采集的激光功率比采用STED低3-5倍。共焦获取过程中的漂白可以忽略不计
    4. 当STED激光波长和荧光团的发射光谱之间存在一些重叠时,发生光子损耗。因此,对于STED通道采集(在共焦获取之后),AF488被激发488nm,光子耗尽592nm,1.2-6nsec HyD门控;罗丹明鬼笔环素用561nm激发,耗尽660nm,1-6nsec HyD门控;并且以647nm激发Atto 647N并用6-6nsec HyD门控耗尽775nm。
    5. 需要注意的是,如果荧光团能够吸收特定波长的能量(即具有包含耗尽激光器的波长的激发光谱的荧光团,592nm, 660nm或775nm)。这是因为在成像平面上的耗尽激光输出比激发源大约500-1,000倍,这将迅速导致光漂白。例如,Atto 647N将通过660nm耗尽激光进行光漂白,Alexa 594将通过592nm耗尽激光器等进行光漂白。因此,为了避免光漂白,STED通道的收集顺序至关重要,首先收集较长波长的荧光团,最后收集较短的波长。在本实验中,STED通道的收集顺序为Atto 647N,其次为罗丹明鬼笔环肽,然后为AF488。
    6. 在惠更斯精华中设置了额外的去卷积参数,以解释由STED光束(STED饱和因子)抑制荧光的预期量,以及光致抗蚀剂对消耗光束(STED immunity fraction)的荧光分子的预期分数。 STED饱和因子是STED激光的绝对强度除以饱和强度。对于最大强度的STED激光,饱和因子为30,并且基于STED激光强度进行缩放。 STED免疫部分是未被STED激光耗尽的荧光团的分数。它描述为添加到纯STED PSF中的附加共聚焦PSF组分,并以最大饱和度的百分比估计。它对解卷积图像的质量影响不大,典型值约为10%。对于RSV F,STED饱和因子的值为30,STED免疫部分为14。对于F-肌动蛋白,这些值分别为27和10。对于微管蛋白,这些值分别为5和11。

笔记

  1. 为了避免在移液时干扰细胞单层,轻轻倾斜板(角度小于45°),并在分配时将移液管尖端朝向每个孔的侧壁引导。
  2. 缓慢移动以保持脆弱的细胞结构和病毒丝。
  3. 为了防止对细胞生物学的可能的干扰作用,培养A549细胞时不要包括抗生素和抗真菌剂
  4. 使用蔗糖纯化的重组RSV不用于这些研究。
  5. 在进行这些研究之前,应通过免疫斑块测定在Vero 76细胞(ATCC CRL-1587)的24孔平板上测定RSV库存的滴度。简而言之,在细胞培养基中制备感兴趣的RSV库存系列十倍稀释液。从亚汇合的Vero细胞单层的24孔板中弃去生长培养基,并将每孔100μl的连续RSV稀释液转移到24孔板的重复孔中。为了允许病毒吸附,在细胞培养箱中孵育2小时;每20分钟轻轻摇动板块,以防止单层干燥。吸附后,每孔用1ml 0.8%甲基纤维素覆盖层(使用细胞培养基制备)覆盖单层,并孵育培养物5-6天。然后通过翻转板丢弃甲基纤维素覆盖物,并用冰冷的80%甲醇固定。通过使用RSV特异性一级抗体制剂(例如市售的针对RSV的小鼠单克隆抗体)免疫染色观察RSV斑块,随后是物种特异性二抗(例如,过氧化物酶缀合的山羊抗小鼠IgG(H + L) ,KPL#074-18064)和选择的检测系统(例如,CN过氧化物酶底物,KPL#50-73-02和过氧化物酶底物溶液B,KPL#50-65-02)。选择产生良好分离的斑块的稀释孔,每孔计数病毒斑块,并通过将平均斑块数乘以稀释因子来计算病毒库存的滴度(每毫升噬斑形成单位)。

数据分析

图像被去卷积,并使用惠更斯反卷积软件进行FWHM测量。使用PRISM软件版本7计算标准偏差。以前在(Mehedi等人,2016)中详细描述了丝状伪足驱动的RSV细胞对细胞扩散的STED显微镜观察。在这里,我们还包括一个额外的通道来显示RSV感染细胞中的微管蛋白网络。

食谱

  1. F-12完整媒介
    500 ml F-12营养组合
    10%FBS
    1%L-谷氨酰胺
  2. 4%PFA
    30 ml 1x DPBS
    10 ml 16%PFA
  3. 0.05%Triton X-100
    50ml 1x DPBS
    250μlTriton X-100
  4. 3%BSA
    50ml 1x DPBS
    1,500μlBSA(1 mg / ml)

致谢

这项研究得到了NIAID,NIH的校内研究计划的支持。所有作者都宣称不存在竞争利益。该协议是从以前发表的工作中采用的(Mehedi等人,2016)。

参考

  1. Bachi,T.和Howe,C。(1973)。< a class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/4128827”target =“_ blank” >呼吸道合胞病毒的形态发生和超微结构 12(5):1173-1180。
  2. Collins,PL,Hill,MG,Camargo,E.,Grosfeld,H.,Chanock,RM and Murphy,BR(1995)。< a class =“ke-insertfile”href =“https://www.ncbi来自克隆cDNA的感染性人呼吸道合胞病毒的生产确认了来自M2的5'近端开放阅读框的转录延伸因子的重要作用。基因表达中的mRNA,并提供疫苗开发的能力。 Proc Natl Acad Sci USA 92(25):11563-11567。
  3. Hell,SW and Wichmann,J。(1994)。通过受激发射来破坏衍射分辨率极限:受激发射 - 耗尽荧光显微镜。选择亮点19(11):780-782。
  4. Le Nouen,C.,Munir,S.,Losq,S.,Winter,CC,McCarty,T.,Stephany,DA,Holmes,KL,Bukreyev,A.,Rabin,RL,Collins,PL and Buchholz,UJ( 2009)。单核细胞衍生的人树突状细胞的感染和成熟人类呼吸道合胞病毒,人类偏肺病毒和人类副流感病毒3型细胞。恶性病毒385(1):169-182。
  5. Mehedi,M.,McCarty,T.,Martin,SE,Le Nouen,C.,Buehler,E.,Chen,YC,Smelkinson,M.,Ganesan,S.,Fischer,ER,Brock,LG,Liang,B 。,Munir,S.,Collins,PL and Buchholz,UJ(2016)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/27926942”目标=“_ blank”>肌动蛋白相关蛋白2(ARP2)和病毒诱导的丝状伪足促进人呼吸道合胞病毒传播。 PLoS Pathog 12(12):e1006062。
  6. Westphal,V.,Rizzoli,SO,Lauterbach,MA,Kamin,D.,Jahn,R。和Hell,SW(2008)。< a class =“ke-insertfile”href =“http:// science。科学 320(5873):246- 249.
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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