Dataset Details
| Title | actin_hela_phalloidin_647 |
|---|---|
| Modality | dSTORM |
| Primary Antibody | phalloidin |
| Primary Antibody Catalog Number | NA |
| Secondary Antibody | |
| Secondary Antibody Catalog Number | |
| Localization Software | ThunderSTORM |
| Fluorophore | alexafluor-647 |
| Protein | actin |
| UniProt Protein ID | |
| Cell Type | HeLa |
| Primary Cell Line | False |
| Cell Line Company | |
| Cell Line Number | |
| DOI | |
| Contact Email/Raw Data Link |
No contact email or raw data link provided. |
| Drift Corrected | False |
| Blink Corrected | False |
| Effective Pixel Size (nm) | 300.0 |
| Experimental Notes | Data provided by Ezra Bruggeman from the Lee lab, University of Cambridge. Localisation software used is POLCAM-SR (https://github.com/ezrabru/POLCAM-SR). Full description of experimental notes: Optical setup: All Experiments were performed on a customized, inverted microscope (Nikon, Eclipse Ti2). Illumination was provided by a 638 nm laser housed in an Omicron LightHUB. The output from the laser was coupled into a single mode optical fibre and collimated into an 9.6 mm diameter beam, using an off-axis parabolic mirror (Thorlabs RC08APC-P01). This beam was focused onto the back focal plane of a 60×, 1.42 NA oil immersion objective (Olympus, PlanApo N) using a 300 mm focal length lens, to achieve wide-field excitation (96 µm FWHM). The sample was illuminated using quasi-TIRF by laterally displacing the excitation beam towards the edge of the back focal plane. The measured power density at the image plane was 3.51 kW/cm^2. A quadband dichroic (Di03-R405/488/561/635-t1, Semrock) was used to seperate fluorescence from the excitation. The emission was filtered using a long-pass filter (BLP01-635R, Semrock) before detection. An exposure time of 30 ms was used. Images were acquired using Micro-Manager 2.0 (using ThorCam dll). Data analysis: Data was analysed using POLCAM-SR (https://github.com/ezrabru/POLCAM-SR). Unprocessed polarisation camera images were converted from analog-to-digital units (ADU) to photons by subtracting a pixel-dependent offset (from a camera calibration) and subsequently dividing by the pixel-dependent gain (from a camera calibration) and quantum efficiency of the camera. The polarisation camera images were then demosaicked using cubic spline interpolation to get the four complete polarised channels. The Stokes parameter images (S0, S1 and S2) were calculated from the four polarised images and localisation was performed on the S0 image. Candidates for localisation were determined by thresholding on a Difference-of-Gaussian filtered S0 image. Localisation candidates were then localized using least-squares fitting of a rotated asymmetric Gaussian. The number of photons was estimated as the integrated volume under the Gaussian fit. The localisation precision was calculated using Eq. (17) in Thompson et al. (https://doi.org/10.1016%2FS0006-3495(02)75618-X). The in-plane angle phi of the emission transition dipole moment of the molecule is estimated as the local weighted average angle of linear polarisation, and the rotational mobility proxy is calculated as the local weighted average of the degree of linear polarisation (weighted averages, where the weights are derived from S0). Sample preparation: Cell culture: HeLa TDS cells were cultured in DMEM (Gibco, Invitrogen) supplemented with 10 % Fetal Bovine Serum (FBS, Life Technologies), 1 % penicillin/streptomycin (Life Technologies), and 1 % glutamine (Life Technologies) at 37 °C + 5 % CO_2. Cells were periodically tested for mycoplasma contamination and passaged 3 times per week. Cells were plated at low density on high-precision glass coverslips (MatTek, P35G-0.170-14-C) 1 day prior to fixation for dSTORM experiments. Labeling: Cells were simultaneously fixed and permeabilized in cytoskeleton buffer (CBS, 10 mM MES, 138 mM KCl, 3 mM MgCl_2, 2 mM EGTA, 4.5 % sucrose w/v, pH 7.4), + 4 % paraformaldehyde (PFA) and 0.2 % Triton for 6 minutes at 37 °C, and further fixed in CBS + 4 % PFA for 14 minutes at 37 °C. Post-fixation, cells were washed x3 in PBST (PBS supplemented with 0.1 % Tween) and permeabilized a second time in PBS + 0.5 % Triton for 5 minutes at room temperature. The samples were then washed 3 times in PBST and blocked for 30 minutes in 5 % BSA. Cells were washed x3 in PBST and then incubated with Alexa Fluor™ 647 Phalloidin (A22287, Invitrogen, 1:50 in PBS) for 1 h in the dark, followed by x2 washes in PBS. Prior to dSTORM imaging, PBS was replaced with dSTORM imaging buffer (base buffer consisting of 0.56 M glucose, 50 mM Tris (pH 8.5), and 10 mM NaCl supplemented with 5 U/mL pyranose oxidase (Sigma, P4234), 10 mM cysteamine (Sigma, 30070), 40 µg/mL catalase (Sigma, C100) and 2 mM cyclooctatetraene (Sigma, 138924). |
| Uploaded by | SanShirg |
| Upload date | 13 Aug 2024, 11:07 a.m. |
| Most Similar Dataset | Tubulin_hela |
| Minimum Dissimilarity Score (thinned) | 0.3980172735967235 |
| Overall Mean Localization Precision (nm) | 23.829 |
Data Files
Self-Similarity Histograms
Self-similarity histograms and statistics for both thinned (left) and unthinned (right) data have been calculated. The dashed red line on both graphs represents the mean.
Help Section
To access our Self-Similarity Interpretation Guide, click here.
Similarity Search Results
Help Section
- To learn more about the similarity algorithm, dissimilarity scores, and the difference between thinned and unthinned data, click here.
- A Dissimilarity Score (thinned) (mean) of "None" indicates that datasets have no grid square densities above the thinning threshold.