Labeling the human respiratory syncytial virus genomic RNA with exogenous probes for fluorescence and electron microscopy

A method for labeling the genomic RNA of the human respiratory syncytial virus, as well as for isolating and examining the labeled filamentous virions was achieved. This method utilized the multiply labeled tetravalent probe design, first described in Santangelo et al. 2009. It was shown that by introducing MTRIPs into RSV infected cells immediately before isolating virus, the genomic RNA within individual filamentous virions could be labeled and imaged. This process did not seem to decrease viral titer or affect viral morphology, and allowed for the imaging of the virus using fixed and live cell conventional fluorescence microscopy and super-resolution microscopic techniques such as dSTORM and STED. The imaging of other structural components of the virus, such as the M protein, and as was discovered, the M2-1 protein was also shown. Additionally, the virus was examined for host proteins of the RLR family, which are involved in the cellular innate immune response. It was found that the protein MDA5 was localized in the isolated filaments. Finally, gold nanoclusters were covalently bound to the RNA probe to create a probe that would generate contrast in cryo-TEM and cryo-ET. By hybridizing the probe to an mRNA encoding GFP, complexing it with a cationic lipid transfection agent, and delivering it to cells before plunge-freezing, it was demonstrated that the mRNA-lipoplex granules could be detected. In conclusion, the method allows for both dynamic and ultrastructural information about the viral genome to be gathered.Ph.D

CW STED nanoscopy with a Ti:Sapphire oscillator

Fluorescence microscopy has become an essential tool to study biological molecules, pathways and events in living cells, tissues and animals. Meanwhile, the conventional optical microscopy is limited by the wavelength of the light. Even the most advanced confocal microscopy or multiphoton microscopy can only yield optical resolution approaching the diffraction limit of ~200 nm. This is still larger than many subcellular structures, which are too small to be resolved in detail. These limitations have driven the development of super-resolution optical imaging methodologies over the past decade. The stimulated emission depletion (STED) microscopy was the first and most direct approach to overcoming the diffraction limit for far-field nanoscopy. Typically, the excitation focus is overlapped by an intense doughnut-shaped spot to instantly de-excite markers from their fluorescent state to the ground state by stimulated emission. This effectively eliminates the periphery of the Point Spread Function (PSF), resulting in a narrower focal region, or super-resolution. Scanning a sharpened spot through the specimen renders images with sub-diffraction resolution. Multi-color STED imaging can present important structural and functional information for protein-protein interaction. In this work, we presented a dual color, synchronization-free STED stimulated emission depletion (STED) microscopy with a Ti:Sapphire oscillator. The excitation wavelengths were 532nm and 635nm, respectively. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. We also imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the super-resolution resolving power over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED A series of biological specimens were imaged with our dual-color STED. © Copyright SPIE

Mirror-enhanced super-resolution microscopy

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of similar to 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.National Instrument Development Special Program [2013YQ03065102]; '973' Major State Basic Research Development Program of China [2011CB809101]; Natural Science Foundation of China [31327901, 61475010, 61428501]; Australian Research Council Centre of Excellence for Nanoscale BioPhotonics [CE140100003]; National Institute of Health [GM094198]SCI(E)PubMed中国科技核心期刊(ISTIC)[email protected]

Achieving λ/10 resolution CW STED nanoscopy with a Ti:Sapphire oscillator

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED. © 2012 Liu et al

Mirror enhanced STED super-resolution microscopy

© 2017 IEEE. Through reflective interference, the axial thickness of confocal point spread function can be easily improved to 100 nm. Six-fold of axial resolution and two-fold of lateral resolution can be obtained for STED nanoscopy

Interactions between poly(A)+ mRNA and cytoskeletal elements in HDF.

<p>(<b>A</b>) β-tubulin, vimentin and phalloidin IF, poly(A)+ mRNA, and PLA between poly(A)+ mRNA and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green) and nuclei (blue) are shown. Single image plane is represented. Inset, images of boxed regions. Scale bar, 10 µm (2 µm in insets). (<b>B</b>) The mean FMTRIP volume was similar (Kruskal-Wallis One Way ANOVA on Ranks, p=0.5) in cells, where the interactions between poly(A)+ mRNA and β-tubulin (n=25, mean=706µm³, s.d.=427µm³), vimentin (n=33, mean=780µm³, s.d.=405µm³), or phalloidin (n=23, mean=916µm³, s.d.=346µm³) were quantified. (<b>C</b>) The mean percentage of FMTRIP colocalized with PLA (PLA-FMTRIP) was significantly different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s014" target="_blank">Table S1</a>) between the interactions of poly(A)+ mRNA with β-tubulin (n=25, mean=2.3%, s.d.=1.5%), vimentin (n=33, mean=11.5%, s.d.=9.7%), or phalloidin (n=23, mean=53.7%, s.d.=19.9%). (<b>D</b>) The mean PLA frequency was significantly different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s015" target="_blank">Table S2</a>) between the interactions of poly(A)+ mRNA with β-tubulin (n=25, mean=0.010µm^-3, s.d.=0.007µm^-3), vimentin (n=33, mean=0.019µm^-3, s.d.=0.016µm^-3), or phalloidin (n=23, mean=0.069µm^-3, s.d.=0.039µm^-3). Error bars, s.d.</p

Characterization of mRNA-Cytoskeleton Interactions <i>In Situ</i> Using FMTRIP and Proximity Ligation

<div><p>Many studies have demonstrated an association between the cytoskeleton and mRNA, as well as the asymmetric distribution of mRNA granules within the cell in response to various signaling events. It is likely that the extensive cytoskeletal network directs mRNA transport and localization, with different cytoskeletal elements having their own specific roles. In order to understand the spatiotemporal changes in the interactions between the mRNA and the cytoskeleton as a response to a stimulus, a technique that can visualize and quantify these changes across a population of cells while capturing cell-to-cell variations is required. Here, we demonstrate a method for imaging and quantifying mRNA-cytoskeleton interactions on a per cell basis with single-interaction sensitivity. Using a proximity ligation assay with flag-tagged multiply-labeled tetravalent RNA imaging probes (FMTRIP), we quantified interactions between mRNAs and β-tubulin, vimentin, or filamentous actin (F-actin) for two different mRNAs, poly(A) + and β-actin mRNA, in two different cell types, A549 cells and human dermal fibroblasts (HDF). We found that the mRNAs interacted predominantly with F-actin (>50% in HDF, >20% in A549 cells), compared to β-tubulin (<5%) and vimentin (11-13%). This likely reflects differences in mRNA management by the two cell types. We then quantified changes in these interactions in response to two perturbations, F-actin depolymerization and arsenite-induced oxidative stress, both of which alter either the cytoskeleton itself and mRNA localization. Both perturbations led to a decrease in poly(A) + mRNA interactions with F-actin and an increase in the interactions with microtubules, in a time dependent manner.</p> </div

Detection of interactions between FMTRIP-hybridized mRNA and cytoskeletal elements using proximity ligation assay (PLA).

<p>(<b>A</b>) Flag (dark green) bound to a neutravidin (yellow) with Cy3B-conjugated (red) oligonucleotides (red dash) formed an FMTRIP. (<b>B</b>) Streptolysin O created entrance for FMTRIP in the cell membrane, allowing access to the mRNA (gray) bound to the β-tubulin (blue), vimentin (magenta), and F-actin (green), via RNA-binding proteins (RBP, brown). (<b>C</b>) Antibodies against the flag (light blue) and the cytoskeletal element (light magenta) in addition to the proximity probes against the antibodies (dark blue and magenta) attached to the FMTRIP-bound mRNA (gray) and the cytoskeleton (green); the oligonucleotides (black dash) on the proximity probes join to synthesize a Cy5-equivalent hybridized DNA product (light green and black dash) via rolling circle amplification.</p

Interactions between β-actin mRNA and cytoskeletal elements in HDF.

<p>(<b>A</b>) β-tubulin, vimentin and phalloidin IF, β-actin mRNA, and PLA between β-actin mRNA and the cytoskeletal elements in HDF were imaged with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), β-actin mRNA (red), PLA (green), and nuclei (blue) are shown. Single image plane is represented. Inset, images of boxed regions. Scale bar, 10 µm (2 µm in insets). (<b>B</b>) The mean FMTRIP volume was similar (Kruskal-Wallis One Way ANOVA on Ranks, p=0.5) in cells, where the interactions between β-actin mRNA and β-tubulin (n=40, mean=567µm³, s.d.=355µm³), vimentin (n=36, mean=607µm³, s.d.=385µm³), or phalloidin (n=30, mean=660µm³, s.d.=389µm³) were quantified. (<b>C</b>) The mean percentage of FMTRIP colocalized with PLA (PLA-FMTRIP) was significantly different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s018" target="_blank">Table S5</a>) between the interactions of β-actin mRNA with β-tubulin (n=40, mean=3.9%, s.d.=3.2%), vimentin (n=36, mean=12.7%, s.d.=7.9%), or phalloidin (n=30, mean=71.5%, s.d.=20.1%). (<b>D</b>) The mean PLA frequency was significantly different (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s019" target="_blank">Table S6</a>) between the interactions of β-actin mRNA with β-tubulin (n=40, mean=0.012µm^-3, s.d.=0.011µm^-3), vimentin (n=36, mean=0.027µm^-3, s.d.=0.023µm^-3), or phalloidin (n=30, mean=0.33µm^-3, s.d.=0.17µm^-3). Error bars, s.d.</p

Arsenite-induced oxidative stress reduced poly(A)+ mRNA binding to F-actin and vimentin, while increasing interactions with β-tubulin.

<p>(A) β-tubulin, vimentin and phalloidin IF, poly(A)+ mRNA, and PLA between poly(A)+ mRNA and the cytoskeletal elements in HDF were imaged at 0 min (or no) and 10 min exposure to arsenite with a laser-scanning confocal microscope. Merged images of the cytoskeleton (white), poly(A)+ mRNA (red), PLA (green), and nuclei (blue) are shown. All image planes are represented. Scale bar, 10 µm. (B) The mean percentage of FMTRIP (PLA-FMTRIP) bound to β-tubulin increased significantly from 0 min exposure (n=28, mean=2.5%, s.d.=1.6%) to 5 min exposure (Kruskal-Wallis One Way ANOVA on Ranks, p<0.001; n=31, mean=12.8%,s.d.=14.6%); remained high for 10 min exposure (n=26, mean=11.3%, s.d.=9.1%); and decreased to the pre-exposure level at 20 (Kruskal-Wallis One Way ANOVA on Ranks, p<0.05; n=22, mean=2.6%, s.d.=3.8%) and 40 min (n=20, mean=2.2%, s.d.=3.0%) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s022" target="_blank">Table S9</a>). (C) The mean percentage of FMTRIP (PLA-FMTRIP) bound to vimentin remained similar to 0 min exposure (n=42, mean=11.1%, s.d.=8.8%) for 5 (n=23, mean=13.9%,s.d.=6.5%),10 (n=19, mean=18.7%, s.d.=7.5%), and 20 min (n=13, mean=13.3%, s.d.=7.1%) exposure; it decreased at 40 min (n=17, mean=5.9%, s.d.=4.4%) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s023" target="_blank">Table S10</a>). (D) The mean percentage of FMTRIP (PLA-FMTRIP) bound to F-actin decreased significantly from 0 min exposure (n=38, mean=49.3%, s.d.=19.4%) to 5 min exposure (Kruskal-Wallis One Way ANOVA on Ranks, p<0.001; n=25, mean=6.0%,s.d.=5.2%); remained low for 10 (n=21, mean=9.6%, s.d.=6.4%), 20 (n=19, mean=15.4%, s.d.=9.4%), and 40 min (n=16, mean=6.8%, s.d.=4.4%) exposure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074598#pone.0074598.s024" target="_blank">Table S11</a>). Error bars, s.d.</p