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6 results for “TIRF”
Example Microscopy Metadata JSON files produced using Micro-Meta App to document the acquisition of example images using a custom-built TIRF Epifluorescence Structured Illumination Microscope
<p><strong>Example Microscopy Metadata JSON files produced using the <a href="https://wu-bimac.github.io/MicroMetaApp.github.io/">Micro-Meta App</a> documenting an example raw-image file acquired using the custom-built TIRF Epifluorescence Structured Illumination Microscope.</strong></p> <p>For this use case, which is presented in Figure 5 of <a href="http://doi: https://doi.org/10.1101/2021.05.31.446382">Rigano et al., 2021</a>, Micro-Meta App was utilized to document:</p> <p>1) The <strong>Hardware Specifications</strong> of the custom build TIRF Epifluorescence Structured light Microscope (TESM; <a href="https://www.pnas.org/content/109/8/E471.long">Navaroli et al., 2010</a>) developed, built on the basis of the based on Olympus IX71 microscope stand, and owned by the Biomedical Imaging Group (http://big.umassmed.edu/) at the Program in Molecular Medicine of the University of Massachusetts Medical School. Because TESM was custom-built the most appropriate documentation level is <strong>Tier 3</strong> (<em>Manufacturing/Technical Development/Full Documentation</em>) as specified by the <a href="https://doi.org/10.5281/zenodo.4710731">4DN-BINA-OME</a> Microscopy Metadata model (<a href="https://doi.org/10.1101/2021.04.25.441198">Hammer et al., 2021</a>).</p> <p>The TESM Hardware Specifications are stored in: <strong>Rigano et al._Figure 5_UseCase_Biomedical Imaging Group_TESM.JSON</strong></p> <p>2) The <strong>Image Acquisition Settings</strong> that were applied to the TESM microscope for the acquisition of an example image (FSWT-6hVirus-10minFIX-stk_4-EPI.tif.ome.tif) obtained by Nicholas Vecchietti and Caterina Strambio-De-Castillia. For this image, TZM-bl human cells were infected with HIV-1 retroviral three-part vector (FSWT+PAX2+pMD2.G). Six hours post-infection cells were fixed for 10 min with 1% formaldehyde in PBS, and permeabilized. Cells were stained with mouse anti-p24 primary antibody followed by DyLight488-anti-Mouse secondary antibody, to detect HIV-1 viral Capsid. In addition, cells were counterstained using rabbit anti-Lamin B1 primary antibody followed by DyLight649-anti-Rabbit secondary antibody, to visualize the nuclear envelope and with DAPI to visualize the nuclear chromosomal DNA.</p> <p>The Image Acquisition Settings used to acquire the FSWT-6hVirus-10minFIX-stk_4-EPI.tif.ome.tif image are stored in: <strong>Rigano et al._Figure 5_UseCase_AS_fswt-6hvirus-10minfix-stk_4-epi.tif.JSON</strong></p> <p><em><strong>Instructional video tutorials on how to use these example data files:</strong></em><br> Use these videos to get started with using Micro-Meta App after downloading the example data files available here.</p> <ul> <li><a href="https://vimeo.com/562022222">Part 1/2</a></li> <li><a href="https://vimeo.com/562022281">Part 2/2</a></li> </ul>
TIRF imaging data of neutrophils migrating underneath endothelial cells
<p>This data is used in the publication "Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling": https://www.frontiersin.org/articles/10.3389/fimmu.2021.667213/full</p> <p> </p> <p><strong>TIRF Microscopy</strong></p> <p>Lentiviral transduction was used to generate an endothelial cell line expressing mNeonGreen-Paxillin (derived from addgene plasmid # 129604). Cells were imaged with a Nikon Ti-E microscope equipped with a motorized TIRF Illuminator unit, a 60x TIRF objective (60x Plan Apo, Oil DIC N2, NA =1.49, WD = 120 um) and Perfect Focus System. Images were acquired with an Andor iXon 897 EMCCD camera and the Nikon NIS elements software. mNeonGreen was imaged using the 488 nm laser line and calcein red-orange was imaged using the 561 nm laser line. A quad split dichroic mirror (405 nm, 488 nm, 561 nm, 640 nm) was used in combination with dual band pass emission filter (515 to 545 nm, 600 to 650 nm). To achieve a larger field of view a 3 x 3 tile scans was acquired with 15% overlap stitching on the GFP channel. Time lapse images were taken every 10 s.</p>
Ent1-mNeonGreen and Abp1-mTq TIRF Raw data from "Sla2 is a core interaction hub for Clathrin Light Chain and the Pan1/End3/Sla1 Complex"
<p>This dataset is raw TIRF movies (as ND2 files) of data shown in Figure 4e of the manuscript "Sla2 is a core interaction hub for Clathrin Light Chain and the Pan1/End3/Sla1 Complex" in BioXiv (https://doi.org/10.1101/2024.11.14.623549) </p> <p><br>In this repository, you will find 20 different movies taken over two microscopy sessions for Ent1-mNeonGreen/Abp1-mTurquoise2 for yeast cells expressing Sla2-WT and two different mutants (called Site1 and Site2 mutants). </p> <p><br>How is it called in the repository / In the manuscript<br>Sla2-WT - Sla2 - WT<br>Sla2-dYYR - Sla2 - ΔSite2<br>Sla2-d515 - Sla2 - ΔSite1</p> <p>Data relating to experimental conditions can be found in https://doi.org/10.1101/2024.11.14.623549</p>
TIRF Microscopy Data Files. "Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides: Implications for target site search"
<p>TIRF-microscopy images and analysis data from single-molecule experiments assessing the direct transfer phenomenon in the TREX1 exonuclease. </p>
High-speed TIRF and 2D super-resolution structured illumination microscopy with large field of view based on fiber optic components
<p>Super-resolved structured illumination microscopy (SR-SIM) is among the most flexible, fast, and least perturbing fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-built instruments are easily able to deliver two-fold resolution enhancement at video-rate frame rates, but the cost of the instruments is still relatively high, and the physical size of the instruments based on the implementation of their optics is still rather large. Here, we present our latest results towards realizing a new generation of compact, cost-efficient, and high-speed SR-SIM instruments. Tight integration of the fiber-based structured illumination microscope capable of multi-color 2D- and TIRF-SIM imaging, allows us to demonstrate SR-SIM with a field of view of up to 150 × 150 μm<sup>2</sup> and imaging rates of up to 44 Hz while maintaining highest spatiotemporal resolution of less than 100 nm. We discuss the overall integration of optics, electronics, and software that allowed us to achieve this, and then present the fiberSIM imaging capabilities by visualizing the intracellular structure of rat liver sinusoidal endothelial cells, in particular by resolving the structure of their trans-cellular nanopores called fenestrations.</p>
TIRF imaging of lysosomal exocytosis
<p><strong><span> </span></strong><strong><span>1\ Training Dataset (120 movies)</span></strong></p> <p><span>This dataset contains the data used for the training and the evaluation of ExoDeepFinder (i.e. the 120 movies presented in figure 1D). Each folder corresponds to a single TIRFM movie of one RPE1 VAMP7-pHluorin transfected cell. All cells are in control conditions <em>i.e. </em>no drug treatment. Importantly, some movies are from paired experiments, before-after treatment, but only conditions before treatment have been included in the dataset. Hence, the mentioning of a drug in the folder name is a reference to the control before the aforementioned treatment and does not indicate drug treatment. While most of the cells are seeded on fibronectin-coated coverslips (62 movies), some of them are seeded on different substrates:</span></p> <p><span>-Cells from folders with the reference “Micropattern Ring” were seeded on fibronectin coated, ring-shaped micropatterns (diameter of 37µm and thickness of the adhesive ring of 7µm) (17 movies).</span></p> <p><span>-Cells from folders with the reference “Rectangles pattern” or “Micropattern rectangle” were seeded on fibronectin coated, rectangle-shaped micropatterns (9x40µm) (18 movies).</span></p> <p><span>-Cells from folders with the reference “PLL coating” were seeded on PLL coated coverslips (23 movies).</span></p> <p><span>Moreover, while most of the cells are only VAMP7-pHluorin transfected (96 movies), some of them have been co-transfected:</span></p> <p><span>-Cells from folders with the reference “Paxillin” were co-transfected with paxillin-mCh (19 movies).</span></p> <p><span>-Cells from folders with the reference “RFP-Rab6A” were co-transfected with mCh-Rab6A (5 movies).</span></p> <p><span>Each folder contains a sequence of .TIF images corresponding to each frame of the movie. Movies can be opened with ImageJ as an image sequence. Each movie is made of 1001 16-bit images. The pixel size is 0.160µm. In addition, each folder contains a .nd file that can be opened with ImageJ. Moreover, a .tif cell mask is associated to each movie (“Flood fill” to fill holes inside the mask and “Clear outside” algorithms are not applied on these masks but necessary to obtain simply connected masks). </span></p> <p><span>Lastly, each folder contains a .txt file with the manual annotations of exocytosis events, considered as the ground truth reference. This .txt file is an output of imageJ. The x,y and t coordinates of exocytosis events are given, respectively, by the columns “X”, “Y” and “Slice”. This file reports x and y coordinates in pixels and time as frame number.</span></p> <p><span>Finally, the dataset contains an excel file (.xlsx) giving for each file:</span></p> <p><span>-Folder name</span></p> <p><span>-The total duration of the movie (based on the actual computer saving times of the frames and not the theoretical time set by the microscope)</span></p> <p><span>-The number of frames</span></p> <p><span>-The invert of the frame rate</span></p> <p><span>-The background <em>i.e.</em> average signal in a region outside the cell at t=1</span></p> <p><span>-The signal <em>i.e. </em>average signal in a region inside the cell at t=1</span></p> <p><span>-The classical SBR, computed as the ratio of the two previous columns</span></p> <p><span>-The exocytosis SBR, computed as the ratio between peak intensity of the event (F) and background before the exocytosis event (F<sub>0</sub>) (see paper methods)</span></p> <p><span>-The SBR level, <em>i.e.</em> the classification as low, medium or high SBR based on the exocytosis SBR rank</span></p> <p><span>-Group (either “training” or “inference”) to specify if the movie has been used for ExoDeepFinder training or evaluation</span></p> <p><span> </span></p> <p><strong><span>2\ Robustness Dataset (90 movies)</span></strong></p> <p><span>This dataset contains the data used for the robustness evaluation (figure 2). It contains 4 sub-datasets. Each sub-dataset is made of several folders containing .TIF images corresponding to each frame of the movie, the corresponding .nd file, a .tif cell mask and a .txt file with the manual annotations of exocytosis events, considered as the ground truth reference. This organization is the same as found in the “Training Dataset”. In addition, each sub-dataset contains an excel file (.xlsx), giving for each folder i) the name, ii) number of frames, iii) the invert of the frame and iv) SBR, similarly to the excel file in the “Training Dataset”.</span></p> <p><span> </span></p> <p><strong><span>2.1\ Bafilomycin Dataset (32 movies)</span></strong></p> <p><span>This dataset corresponds to RPE1 cells transfected with VAMP7-pHluorin, seeded on fibronectin-coated coverslips and treated with Bafilomycin A1 (at 100nM for 1h). Each cell is imaged before and 1h after the treatment. The following table shows the pairing.</span></p> <table> <tbody> <tr> <td> <p><strong><span>Before Bafilomycin A1</span></strong></p> </td> <td> <p><strong><span>After Bafilomycin A1</span></strong></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell1avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell1apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell2avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell2apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell3avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell3apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell4avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell4apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell5avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell5apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell6avant</span></p> </td> <td> <p><span>TIRF 191204 VAMP7 +Bafalomycine A_Cell6apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell1avant</span></p> </td> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell1apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell2avant</span></p> </td> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell2apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell3avant</span></p> </td> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell3apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell4avant</span></p> </td> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell4apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell5avant</span></p> </td> <td> <p><span>TIRF 211118 VAMP7 + Bafilomycine A_Batch1_Cell5apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell1avant</span></p> </td> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell1apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell2avant</span></p> </td> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell2apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell3avant</span></p> </td> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell3apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell4avant</span></p> </td> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell4apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell5vant</span></p> </td> <td> <p><span>TIRF 211223 VAMP7 + Bafilomycin 3_Batch1_Cell5apres</span></p> </td> </tr> </tbody> </table> <p><span> </span></p> <p><strong><span>2.2\ Histamine Dataset (34 movies)</span></strong></p> <p><span>This dataset corresponds to RPE1 cells transfected with VAMP7-pHluorin, seeded on fibronectin-coated coverslips and treated with histamine (at 100µM). Each cell is imaged before and immediately after the treatment. The following table shows the pairing.</span></p> <table> <tbody> <tr> <td> <p><strong><span>Before Histamine</span></strong></p> </td> <td> <p><strong><span>After Histamine</span></strong></p> </td> </tr> <tr> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell1AvantStimulation</span></p> </td> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell1ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell2AvantStimulation</span></p> </td> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell2ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell3ApresStimulation</span></p> </td> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell3Apres2Stimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell4AvantStimulation</span></p> </td> <td> <p><span>TIRF 190516 VAMP7 + Histamine_Cell4ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell1AvantStimulation</span></p> </td> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell1ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell2AvantStimulation</span></p> </td> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell2ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell3AvantStimulation</span></p> </td> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell3ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell4AvantStimulation</span></p> </td> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell4ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell6AvantStimulation</span></p> </td> <td> <p><span>TIRF 190517 VAMP7 + Histamine (2 replicat)_Batch1_Cell6ApresStimulation</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell1avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell1apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell2avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell2apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell3avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell3apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell4avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell4apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell5avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell5apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell6avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell6apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell7avant</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell7apres</span></p> </td> </tr> <tr> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell8avant1</span></p> </td> <td> <p><span>TIRF 220121 VAMP7 + Histamine 3_Batch1_Cell8avant</span></p> </td> </tr> </tbody> </table> <p><span> </span></p> <p><strong><span>2.3\ HeLa Dataset (14 movies)</span></strong></p> <p><span>This dataset corresponds to HeLa cells transfected with VAMP7-pHluorin and seeded on fibronectin-coated coverslips.</span></p> <p><span> </span></p> <p><strong><span>2.4\ CD63 Dataset (10 movies)</span></strong></p> <p><span>This dataset corresponds to RPE1 cells transfected with CD63-pHluorin and seeded on fibronectin-coated coverslips.</span></p> <p><span> </span></p>
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