Community-developed checklists for publishing images and image analysis

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality of microscopy data is in publications.Comment: 28 pages, 8 Figures, 3 Supplmentary Figures, Manuscript, Essential recommendations for publication of microscopy image dat

Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells.

Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells

Detection of proximity between NP-mCherry and vRNPs in infected cells by proximity ligation assay.

<p>The anti-NP monoclonal antibody 3/1 and a rabbit antibody recognizing mCherry were used. Maximal intensity projections of the z-stacks acquired with a laser scanning confocal microscope are shown. Scale bar: 10 μm. Pseudocolors: white, PLA signal; red, mCherry; blue, nuclei staining (DAPI).</p

Fluorescence correlation spectroscopy data for PB2-GFP_comp- and NP-mCherry-labeled species in the nuclei of HEK-293T cells.

<p>Fluorescence correlation spectroscopy data for PB2-GFP_comp- and NP-mCherry-labeled species in the nuclei of HEK-293T cells.</p

Colocalization of transiently expressed NP-mCherry with vRNPs in infected A549 cells.

<p>Mock-infected cells (top panels) or cells infected with the WSN-wt influenza virus (bottom panels) are shown. Cells were fixed at 6 hpi and stained with the anti-NP monoclonal antibody clone 3/1. Scale bar: 10 μm. Pseudocolors: red, mCherry; green, NP; blue, nuclei staining (DAPI).</p

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation

FRET efficiency between PB2-GFP_comp and NP-mCherry in the nuclei of HEK-293T cells.

<p>FRET efficiency between PB2-GFP_comp and NP-mCherry in the nuclei of HEK-293T cells.</p

Fluorescence correlation spectroscopy data for GFP_comp-labeled species (green) and mCherry-labeled species (red).

<p>The experimental autocorrelation data, fitted curves (in black), and residuals, are shown for an individual representative HEK-293T cell. A. Control cell transiently expressing MBD-GFP_comp and mCherry and infected with the WSN-wt virus; single-component translational diffusion model. Arrow points to the inflection point of the autocorrelation curve. B. Cell transfected with GFP1-10 and NP-mCherry and infected with the WSN-PB2-GFP11 virus. Two-component translational diffusion model.</p

FLIM-FRET microscopy in live HEK-293T cells.

<p>A. The principle of FLIM-FRET assay with GFP_comp and mCherry as the FRET donor and acceptor, respectively, is schematically drawn; a representative fluorescence decay dataset fitted curve (in red) and residuals for a single pixel within the nucleus of a cell infected with the WSN-PB2-GFP11 virus are shown. B. Fluorescence intensity (left and middle panels) and mean GFP_comp fluorescence lifetime (right panels) images of the infected and/or transfected cells. Graphs to the right of the micrographs show the distributions of mean GFP_comp fluorescence lifetime values (occurrence of pixels with a given mean lifetime) in the nuclei pointed by yellow arrowheads in GFP_comp intensity images (middle right panels). Sketches on the far right show the “observable”, fluorescently labeled species for each sample; virus ideographs indicate viral infection, either with WSN-wt (in positive and negative controls) or with WSN-PB2-GFP11 (in other infected samples).</p