DOI: 10.5061/dryad.xsj3tx9cn
title: Comparing Lifeact and Phalloidin for super-resolution imaging of actin in fixed cells

description: Visualizing actin filaments in fixed cells is of great interest
 for a variety of topics in cell biology such as cell division,
 cell movement, and cell signaling. We investigated the
 possibility of replacing phalloidin, the standard reagent for
 super-resolution imaging of F-actin in fixed cells, with the
 actin binding peptide `lifeact’. We compared the labels for use
 in single molecule based super-resolution microscopy, where
 AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality
 and Atto 655 labeled lifeact was used in a single molecule
 imaging, reversible binding modality. We found that imaging with
 lifeact had a comparable resolution in reconstructed images and
 provided several advantages over phalloidin including lower
 costs, the ability to image multiple regions of interest on a
 coverslip without degradation, simplified
 sequential super-resolution imaging, and more continuous labeling
 of thin filaments.
description: Actin structures of HeLa and RBL cells were labeled in two
 approaches, lifeact-Atto655 and phalloidin-AF647. Data was collected for
 each method using single-molecule localization microscopy. The shared data
 include the raw sequences taken for each condition for various number of
 samples in both HeLa and RBL cells as named. To evaluate the capability of
 labeling approaches in sequential super-resolution imaging, actin
 structures and microtubules were labeled in HeLa cells for each
 treatment.   To create figure 1
 & 3 super-resolution images:
 LifeactApproach-HeLaCell-Sample1.h5
 PhalloidinApproach-HeLaCell-Sample1.h5
 LifeactApproach-RBLCell-Sample1.h5
 PhalloidinApproach-RBLCell-Sample1.h5
   To create figure 2 FRC
 calculation:
 LifeactApproach-HeLaCell-Sample1.h5
 LifeactApproach-HeLaCell-Sample2.h5
 LifeactApproach-HeLaCell-Sample3.h5
 PhalloidinApproach-HeLaCell-Sample1.h5
 PhalloidinApproach-HeLaCell-Sample2.h5
 PhalloidinApproach-HeLaCell-Sample3.h5
 LifeactApproach-RBLCell-Sample1.h5
 LifeactApproach-RBLCell-Sample2.h5
 LifeactApproach-RBLCell-Sample3.h5
 PhalloidinApproach-RBLCell-Sample1.h5
 PhalloidinApproach-RBLCell-Sample2.h5
 PhalloidinApproach-RBLCell-Sample3.h5
   To create figure 4 multiple dataset
 collection:
 LifeactApproach-HeLaCell-Sample4.h5
 LifeactApproach-HeLaCell-Sample5.h5
 PhalloidinApproach-HeLaCell-Sample3.h5
 PhalloidinApproach-HeLaCell-Sample4.h5
   To create figure 5 sequential
 super-resolution imaging:
 LifeactApproach-HeLaCell-Sample6.h5
 LifeactApproach-HeLaCell-Sample6-Microtubules.h5
 PhalloidinApproach-HeLaCell-Sample6.h5
 PhalloidinApproach-HeLaCell-Sample6-Microtubules.h5
   HDF5 files can be loaded into MATLAB.  As an
 example, to access the images for sample 1, run the
 following commands in MATLAB:   1)
 Find the number of sequences for a sample:  
    Info =
 h5info('LifeactApproach-HeLaCell-Sample1.h5')
   2) Each level of the h5 file hierarchy is
 named in the 'Groups' structure. For the data
 included here, we have two layers: /Channel01/Zposition001/.
 To find the number of the sequence:
      Num_Dataset =
 numel(Info.Groups.Groups.Datasets)  
 3) To load a data set:     
 DataSet_1 =
 h5read('RawData\LifeactApproach-HeLaCell-Sample1.h5',
 ...                      
 '/Channel01/Zposition001/Data0001');
description: All related notes are included in the methods section above or in
 README files included with files in the collection.