u2os cells expressing galectin3 mcherry  (ATCC)

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: 3D Tomogram of the SIM and X-Ray Data Used to Assess Correlation Error Estimation, Related to Figure S3. Z stack motion through a U2OS cells with red fluorescent mitochondria and green fluorescent gold nanoparticles (diameter of 150 nm). Fluorescence and X-ray absorption signal were superimposed using rigid 3D alignment with eC-CLEM as described in the Results section.

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques:

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: Design and performance of the super-resolution fluorescence microscope CryoSIM (A and B) Widefield (WF) (A) and SIM lateral point spread (B) of a 175 nm diameter, 505/515 nm wavelength microsphere (PS-Speck Thermo Fischer Scientific) collected on the cryoSIM at 71 K by using 488 nm light for excitation and a 520/35 nm filter (Semrock) for emission. (C) Plot of lateral point spread (single line profile) of (A) and (B) and their respective Gaussian fits showing clear resolution enhancement. Analysis of representative cryo-SIM data from a single mammalian U2OS cell containing structures tagged with mCherry. (D) Z axis projection of the raw SIM data processed to produce a widefield image stack. (E) Z axis projection of the same data deconvolved with a standard Richardson-Lucy iterative deconvolution algorithm. (F) SIM data fully reconstructed showing the resulting resolution enhancement. (G) Fourier information content analysis of (H)–(K) (flattened and sampled radially) showing the relative increase in information content after reconstruction. The vertical blue bars show the resolution achieved at 525 nm emission in widefield (420 nm, 1/2.38) and SIM (200 nm, 1/5). (H and I) Reciprocal space resolution plots of the widefield data in the cell shown in (D) (Alexa488 and mCherry signal, respectively). (J and K) Same data as (H) and (I), respectively, from the SIM reconstruction shown in (F). Comparing (J) with (H) and (K) with (I) shows the increased image resolution in SIM by the extra intensity further from the origin and therefore at higher frequencies. Concentric dashed circles denote resolution boundaries of 600 nm (small circle) and 200 nm (large circle). All image analysis was done using Fiji ( Schindelin et al., 2012 ) and SIMcheck ( Ball et al., 2015 ). (L) SPEKcheck ( Phillips et al., 2018 ) display of the cryoSIM for a representative fluorophore (Alexa488) excited in the system with an efficiency of 69% and a resulting collection of 70% of the total emitted light. Laser light is shown as a bright blue spike, excitation and emission are shown in shades of light green, and the total transmitted light is shown in bright green. The system dichroic mirrors and filters are also represented (Abbreviations are as follows: ZT, system dichroics; T560, detector splitter dichroic; EM, emission filter).

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Fluorescence, Microscopy

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: CryoSIM resolution parameters, related to Figure 1 Line scans laterally (A and C) and axially (B and D) through single fluorescent beads at 525 nm (A and B) and 605 nm (C and D) emission in widefield and SIM images. Widefield data presented as circles and SIM as diamonds with Gaussians fits as lines. In (E–H) are parts of a raw SIM image of fluorescent mitochondria with decreasing stripe width. 2nd order stripes are 406, 361, 325 and 295 nm respectively. In (I) are line scans through Fourier transforms of the data in (E–H), with varying SIM illumination stripe widths, showing the peaks due to the first order stripes (around 1–1.8 μm −1 ) and second order (around 2.4 to 3.5 μm −1 ). As stripe width decreases, second order peaks decrease in amplitude and shift along the y axis. The black arrow indicates the stripe width, 396 nm, used for the experimental data in the paper. Representative brightfield image of vitrified U2OS cells (J), before (pre-exposure) and (K), after (post-exposure) seven cycles of cryoSIM imaging of green, red, and far red intracellular fluorophores (525 nm, 605 nm and 647 nm) showing that there is no localized ‘melting’ due to exposure to laser light for SIM purposes. In (L) and (M), are orthoslices of (J) and (K), respectively. SIM imaging from the last round of data collection shows the preserved cellular features: (N), green fluorescent lipid droplets, (O), red fluorescent endoplasmic reticulum and (P), far-red fluorescent mitochondria corresponding to the brightfield data shown in (K).

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Imaging

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: X-ray absorption data contrast in cells using different TXM objectives, related to Figure 2 (A) X-ray projection of a 16x16 μm FOV collected at the beamline B24 TXM using the 40 nm objective in a U2OS mammalian cell sample 1h after it has been exposed to infectious reovirus at MOI of 50. The zoomed view in square at the bottom left of the image is located outside the cell. The backing surface (Quantifoil™) can be identified by the regular pattern of circles (needed for blotting prior to vitrification). (B) The diagonal dotted line defines the line used to generate (B), a line profile of pixel intensity in Fiji ( Schindelin et al., 2012 ). Dips in the profile correspond to features of high X-ray absorbance such as membranes and organelles (corresponding vertical dotted lines have been added as examples). We expect reovirus particles to be present both in and outside the cell but given the high background it is be difficult to unambiguously identify. (C and D) Closeups of marked areas showing contrast that could be attributed to reovirus particles. (E) The associated line profile, clearly contains peaks that are 4-5 pixels apart; at 16nm per pixel that gives an object diameter of approximately 80 nm which matches the expected diameter of a reovirus particle. However, the ratio of pixel values is at best 1.1:1.3 even in this ‘ideal’ area (low background without any overlap with cellular content) making the unambiguous identification of individual virus impossible in this sample. (F) X-ray projection of a 10x10 μm FOV collected at the B24 TXM using the 25 nm objective in the cytoplasm of a BSC-1 cell 16 h after it has been exposed to infectious reovirus at MOI of 50-100. The observed regular pattern of small dots is indicative of viral factories with each of these dots representing a single viral particle. (G) A close up of the boxed area in (F), with a line drawn to generate (H), a line profile of pixel intensities. Full width half maxima of this plot give an average diameter of 60nm for these particles which appear to be an array of immature particles in the viral factory having yet to assemble an outer capsid before cell exit.

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Virus

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: Tracking reovirus endosomal trafficking and escape (A) Confocal images of U2OS cells expressing late-endosome marker Rab7-eGFP infected with Alexa647-labeled reovirus at indicated times AI. (B and C) Mammalian reovirus (MRV) presence in late endosomes up to 4 h AI (B). Numbers of Gal3--positive endosomes containing MRV for the same period (C) (error bars indicate SD of n = 8 in B and n = 9 in C). (D–K) CryoSIM images of MRV labeled with Alexa488 in mCherry-Gal3 expressing cells showing the concentrations of Gal3 signal in distinct vesicles in red; brightfield and SIM slices respectively at: time 0 mock-uninfected (D and E); 1 h AI (F and G); 2 h AI (H and I); and 4 h AI (J and K). (L) Superposition of SIM processed data of a 4 h AI sample on the 2D X-ray mosaic of the same area and expansion of adjacent FOVs where two adjacent X-ray tomograms were collected and stitched. (M) Expanded region of interest (highlighted purple in L). (N and O) Same as (M) with Gal3 signal in red (N) and reovirus signal in green (O), respectively. (P–R) ROI from 2 h AI with either X-ray grayscale imaging alone or superposed wiith fuorescence signal: single z axis slice from the corresponding tomogram with Gal3 (red) and MRV (green) localization. Multi-vesicular bodies (MVBs) are clearly identifiable with distinct sub-compartments where Gal3 is concentrated (presumably the result of virus-carrying vesicles fusing with virus free late endosomes). Virus-induced carbon-rich substructures are marked by yellow arrows and vesicle areas within MVBs that remain impermeable to Gal3 marked by blue arrows. (S) Overview of the relevant 2 h AI region with both fluorescent signals overlaid.

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Expressing, Marker, Infection, Labeling, Imaging, Virus

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: SXT and SIM imaging of mock-infected and reovirus-infected U2OS adherent cells, related to Figure 6 (A–C) Representative cross-sections from three cells, (A), (B), and (C), in the control population of U2OS cells. Cellular structures are identified with arrows: pink for the gold nanoparticle fiducials (250 nm), purple for mitochondria, red for the nuclear envelope, orange for the endoplasmic reticulum, green for MVBs, light blue for endo/lysosomes, gray for nucleoli. (D) 2D X-ray mosaics of a grid area with reovirus mock-infected U2OS cells with the corresponding SIM fluorescence slice aligned. The areas where higher resolution X-ray data were collected are outlined in blue (two ROIs ) and expanded. (E) Areas of focus in single slices from the 3D tomograms are shown as four overlapping frames with (from left to right): X-ray absorption only, composite of X-ray absorption and red fluorescence, composite of X-ray absorption and green fluorescence and composite of X-ray absorption with both fluorescence signals. (F and G), same as (D) and (E) respectively but for a cell population 1 h after infection. (H and I), same as (D) and (E) respectively but for a cell population 2 h after infection. (J and K), same as (D) and (E) respectively but for a cell population 4 h after infection. Black bars are 1 μm and white bars 10 μm. In (E), spurious signals represent background levels of fluorescence in U2OS cells; both green and red signals are perfectly co-localized, weak (relative to those observed later in the infection) and are not associated with any distinct cytoplasmic structures. In (G), 1h after infection, a number of small but distinct vesicles are both Gal3 and MRV positive while in (I), smaller endosomes have now merged to give rise to MVBs that have distinct compartments infused with Gal3 while others have not been similarly compromised (presumably arising from concatenation of membranes from MRV-free vesicles). Carbon-rich virus-induced domed structures are also seen in single vesicles. In (K), fluorescence is shown in smaller vesicles alongside large vesicle superstructures shown in Figure 6 M.

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Imaging, Infection, Control, Fluorescence, Virus

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet: Correlation of X-ray and fluorescence data in three dimensions using the beamline B24 platform All the data shown here are from a U2OS cell vitrified 4 h AI with high titers of reovirus T3D. Viral components are fluorescently labeled with Alexa488 (green fluorescence), and there is also an endogenously expressed reporter molecule (Galectin-3) that is accumulated in vesicles carrying concentrated reovirus outer capsids and that carries the mCherry fluorophore (red fluorescence). (A) Z slice from the middle of a soft X-ray absorption tomogram (from beamline B24 using the 40 nm objective) of a U2OS cell with the nucleus on the right side of the figure (red arrow; the two leaves of the nuclear membrane are seen clearly) and the cytoplasmic area with a substantial multi-vesicular body/endolysosome (pale blue arrow), containing folded carbon-dense material given the increased X-ray absorption in that structure (pale yellow arrow). In the thinner areas of the distal cytoplasmic region, several holes can be seen as part of the support film used for cell attachment (deep purple arrows). (B) Ortho-slices of (A), showing a representative portion of the volumetric data. (C) 3D rendering of the cryoSIM recorded structures displaying green fluorescence within and around the endolysosome. (D) 3D semi-transparent rendering of all cytoplasmic vesicles that contain red Galectin-3 fluorescence (data also collected on the cryoSIM) and are seen as an indication of viral presence in those vesicles. All images were generated with Chimera ( Pettersen et al., 2004 ).

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Fluorescence, Labeling, Membrane, Cell Attachment Assay, Generated

Journal: Cell

Article Title: 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

doi: 10.1016/j.cell.2020.05.051

Figure Lengend Snippet:

Article Snippet: U2OS cells (ATCC), or U2OS cells expressing galectin3-mCherry (a kind gift from Harold Wodrich, Bordeaux) were kept in Dulbeccos’ modified Eagle medium (DMEM) (Thermo Fischer Scientific) containing 10% Fetal Bovine Serum (FBS) (Capricorn) and 1% vol/vol of penicillin/streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO 2 .

Techniques: Virus, Recombinant, Modification, Infection, Expressing, Plasmid Preparation, Software, Microscopy