Fig 1.
High-throughput imaging of influenza using super-resolution microscopy.
A) Schematic of the labelling protocol. Virus samples were dried directly onto glass coverslips pre-coated with poly-L-lysine before being fixed, permeabilised and stained with antibodies using a standard immunofluorescence protocol. B) A representative field of view (FOV) of a widefield image of labelled A/Udorn/72 influenza, imaged in the green channel. Scale bar 10 μm. C) A representative FOV of a widefield image of a virus negative sample, imaged in the green channel. Scale bar 10 μm. D) The corresponding dSTORM image of the FOV in B), where HA is labelled in green and NA is labelled in red. Scale bar 10 μm. E-G) Zoomed in images from D) showing individual filaments and spherical particles. Scale bar 5 μm.
Fig 2.
Widefield imaging and automated length analysis of A/Udorn/72 reveals that filaments fit a single distribution.
A) A pipeline for analyzing filamentous Udorn particles uses multiple widefield images, which are adjusted and binarised before being skeletonized to get a measure of filament length. Scale bar 5 μm. B, C&D) Zoomed in skeletons from A). E) Widefield images of A/Udorn/72 virus stained with an antibody against the HA protein were analysed using the pipeline in A). The resulting frequency distribution of the lengths of 46,872 filamentous particles, fit with a double exponential (red line; equation above plot). F) Histogram from the analysis of 243 virus-negative FOVs demonstrates that the background was negligible. G) Zoomed in histogram of F).
Fig 3.
Super-resolution imaging and size analysis of spherical virus particles.
A) A pipeline for analyzing spherical and bacciliform particles using multiple super-resolution images. Super-resolution localisations are clustered using DBSCAN and each cluster is fitted with an ellipse. Particle dimensions are extracted using the major and minor diameters of each ellipse. Scale bars 1 μm. B) A histogram of the major axis lengths shows that influenza virions stained for the HA protein fall into two distinct populations, centered at 75.1nm and 162.4 nm. C) Representative particles from the population centered at 75.1 nm. Scale bar 100 nm. D) Representative particles from the population centered at 162.4 nm. Scale bar 100 nm. E) Histogram of the major/minor axis ratio of influenza particles shows a single distribution. F) Representative super-resolution images of SARS-CoV-2 virions dual-labelled with anti-spike and anti-nucleocapsid primary antibodies and secondary antibodies labelled with Alexa647 (red) and Alexa546 (green) respectively. Scale bar 100 nm. G) A histogram of the spike protein major axis lengths fitted with a Gaussian function shows that SARS-CoV-2 virions fall into a single population, centered at 94.0 nm. H) Analysis of the nucleocapsid protein also falls into a single population centered at 81.8 nm.
Fig 4.
Analysis of the frequency distribution of influenza surface proteins.
A&B) Representative super-resolution images of filaments labelled with A) HA antibody and B) NA antibody. Scale bars 1 μm. C) A pipeline for analyzing influenza filament surface protein distribution using multiple super-resolution images. Super-resolution images are skeletonized, fitted with quadratics along the skeletons, the normal is found before the intensity across this normal is summed, and finally a Fourier transform is taken and summed across filaments. D) The frequency distribution of the HA proteins from 1,067 filaments from 8 FOVs. The frequency spectrum for HA has no distinctive peaks, suggesting that there is no common spatial patterning of HA across filaments. E) The frequency spectrum for NA also has no distinctive peaks, suggesting that there is no common spatial patterning of NA across filaments.
Fig 5.
Filamentous virion simulations and analysis of alternation on simulated dSTORM images.
A&B) Representative simulated dSTORM images of filamentous influenza particles with A) random protein placement and B) 0.01 nm-1 frequency alternating protein placement. HA is shown as green and NA as red, on a filament of 500 nm in length and 80 nm in width. Scale bars 50 nm. C) Intensity profile for a representative simulated filament with randomly distributed HA and NA. D) Same as C) but for filaments with alternating HA/NA. E) The sum of the Fourier transforms of the intensity plots of 1000 simulated filaments with randomly distributed HA and NA, suggesting no common patterning exists. F) Same as E) but for filaments with alternating HA/NA. The peak at 0.01nm-1 and harmonic at 0.02nm-1 confirm that protein patterning can be detected using this method.
Fig 6.
NA is enriched at the tip of budding influenza filaments.
A) Diffraction limited images of filamentous A/Udorn/72 stained with for HA (green; left) and NA (red; right) budding out of infected cells. Scale bars 10 μm. B) Zoomed in merged image of filament highlighted in white box in A). Scale bar 1 μm. C) Normalised averaged intensity traces of the HA signal from 34 filaments showing a uniformly distributed distribution along the filament length. D) Normalised averaged intensity traces of the NA signal showing a ~2-fold enrichment at the filament ends furthest from the cell membrane.
Fig 7.
RNA is located at one end of influenza filaments.
A&B) Diffraction limited images of filamentous A/Udorn/72 stained with an anti-HA antibody (green) overlaid with super-resolution localisations from an array of FISH probes against the NA gene segment (red). Scale bars 1 μm. White boxes denote Archetti bodies. C) Top: Raw intensity traces (grey) of the HA signal from 68 filaments, with the average intensity profile shown as a green line. Bottom: Normalised average HA intensity from 68 filaments. D) Same as C) but for the red super-resolution signal corresponding to NA RNA from the 68 filaments. E) Top: Raw intensity traces (grey) of the RNA signal from 14 filaments with a visible Archetti body, with the average intensity profile shown as a green line. Bottom: Normalised average RNA intensity from the 14 filaments. F) Same as E) but for the RNA signal from 54 filaments with no visible Archetti body.