DOI: 10.5281/zenodo.7620025
title: STORM Vectashield datasets (Tubulin)

description: Data generated at EPFL (LEB) under the supervision of Suliana Manley. Microscope: IX71 (Olympus) with 100x 1.3NA objective (Olympus, UplanFL) mounted on a piezo objective scanner (P-725 PIFOC, Physik Instrumente) +1.6x magnification lens (except Fig1 dataset, 160nm px) Laser: 641 nm (Coherent, CUBE 640–100C) and 561 nm (Coherent Sapphire) (Fig7, Cy3) Intensity on the sample ≈ 1–2 kW/cm<sup>2</sup> Filters: multiband dichroic (89100bs, Chroma) + bandpass emission filter (ET700/75, ET600/75 (Cy3) Chroma), Camera: IxonEM+ (Andor) with an effective 100 nm pixel size and using the conventional CCD amplifier (except when specified, check the _readme.txt files in each dataset) See spec sheet ("Spec-Sheet_iXon.JPG") "FigX" datasets were used to generated the figures of the article "Simple Buffer for 3D STORM Microscopy" (https://doi.org/10.1364/BOE.4.000885): Fig1: STORM Imaging of Alexa647 in pure Vectashield (px 160nm) Fig4: STORM Imaging of Alexa647 in 25% and 50% Vectashield in Glycerol-TRIS Fig5: STORM Imaging of Alexa647 in 25% Vectashield - 75% TRIS-Glycerol + 1% NPG / 20 mM DABCO /10 mM Lipoic Acid Fig6: 3D-STORM Imaging of Alexa647 in 20% Vectashield - 80% TRIS-Glycerol (astigmatism) Fig:7: STORM Imaging of Alexa647 in 25% Vectashield - 75% TDE and 50% Vectashield- 50% PBS Fig8: STORM Imaging of CEP152-Cy3 in 40% Vectashield + 1% NPG + 20 mM DABCO Fig9: STORM Imaging of CF647 and Cy5 in Vectashield HD datasets: 2D (& one 3D) high density datasets, one of which ("2DHD6") was used for the 2013 IEEE ISBI SMLM challenge, while another ('2DHD_3") was used in "FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data" (https://doi.org/10.1038/srep04577) "Vecta_20pc_1" was used to generate Fig S13 in "FALCON: fast and unbiased reconstruction of high-density super-resolution microscopy data" (https://doi.org/10.1038/srep04577) - similar conditions and samples as with High Density but with Low Density. "_STD' files correspond to the standard deviation of the raw datasets. "_STORM" files correspond to STORM reconstructions obtained with <em>Detection of Molecules (DoM) plugin for ImageJ (https://github.com/UU-cellbiology/DoM_Utrecht).</em>" Sample preparation: Cell Culture: COS-7 were cultured in DMEM 10% FBS and plated on Ethanol-cleaned 25 mm size 1 cover-glass (Menzell) or 25mm Hestzig cover-glass (with embedded gold colloids fiducials) Fixation: Cells were pre-extracted for 10s in 0.5% Triton X-100 (Triton) in BRB80 (80 mM PIPES, 1 mM MgCl<sub>2</sub>, 1 mM EGTA, adjusted to pH 6.8 with KOH) supplemented with 4 mM EGTA, washed in PBS, fixed for 10 min in − 20°C-Methanol and washed again in PBS. Immunostaining (all done at room temperature): Blocking 30 min in 5% BSA, then incubation 1.5h with 1:1000 primary antibody in PBS - 1% BSA - 0.2% Triton (PBST), 3 washes with PBS-0.2% Triton, then incubation 45min in PBST with 1:1000 secondary antibody and 3 washes with PBS-0.2% Triton. Primary antibody (except when specified in readme.txt file): mouse anti alpha-tubulin (Sigma-Aldrich, T5168) Secondary antibody (except when specified in readme.txt file): goat anti-mouse Alexa-647 F(ab)2 secondary antibody fragments (Life Technologies, A-21237).
description: Acknowledgements: -Suliana Manley & Pierre Gönczy (Funding & Supervision) -Lina Carlini, Sasha Benke, Julia Gunzenhäuser, Thomas Pengo, Carine Ben Adiba, Nicolas Piro, Vinoth Sundar Rajan, Hélène Perreten, Seamus Holden. (Help with cell culture, data acquisition, data analysis, and discussions)