This package includes the Matlab code for analyzing the dataset recorded on the 4Pi-SMS microscope.
- Microsoft Windows 7 or newer, 64-bit
- CUDA capable graphics card, minimum Compute Capability 3.0
- CUDA 8 compatible graphics driver (for GeForce products 378.66 or later)
- Matlab R2017b or newer
- Curve Fitting Toolbox
- Optimization Toolbox
- DIPimage Toolbox (http://www.diplib.org/download)
- Some files from SMAP (included in this package) (https://github.com/jries/SMAP)
- Download and install Matlab of the right version
- Download and install DIPimage
- Download the code to your computer
- Download example dataset
- Start Matlab and run the code
An example dataset of immunolabeled microtubules of a COS-7 cell is provided.
Download the dataset from: https://drive.google.com/open?id=1I3vBu9UyKaMJ6h1eGCE8DSasIcNGrG9X
Additonal information of the dataset
- Cell images: 20 cycles included (3000 frames per cycle)
- Labeling: anti-a-tubulin primary antibody and CF660C conjugated secondary antibody
- Imaging conditions: 100 fps with a 642 nm laser at 7.5 kW/cm2
- Images of fluorescence beads for channel alignment (10 frames)
- Images of fluorescence beads with 20 nm step sizes for phase shift estimation (61 steps)
- Start Matlab program
- In Matlab, run "dipstart" to initialize DIPimage toolbox
- In Matlab, run "SMS-4Pi" to open the GUI interface
- Click "Main Folder" and select the folder of the example dataset
- Generate calibration file 1
- On the GUI Menu, click "Channel Alignment" -> "Load", select the file in the folder "Beads_align"
- A calibration file named "align_642_FMTtransform_datestring.mat" will be generated
- Generate calibration file 2
- On the GUI Menu, click "Find Phaseshift" -> "Load", select all the file in the folder "Beads_stack"
- Two calibration files named "bead_642_Astfit_datestring.mat" and "bead_642_dphi_cali_datestring.mat" will be generated
- Step 1: get positions of the single molecules
- On the GUI file dispaly window, select the example dataset folder "\4Pi-SMS-Example-Dataset\Cell04", click "Get Positions"
- A result file named "Cell04_642_tmpresult_datestring.mat" will be genrated
- Step 2: phase unwrapping, drift correction and stitching
- Click radio button "Reconstruct" to switch the GUI display
- On the GUI file display window, select "Cell04_642_tmpresult_datestring.mat", click "Reconstruction"
- Step 3: visualization
- A result file named "Cell04_642v20_60.mat" will be generated, which includes 3D positions of localized molecules and can be loaded in PYME for visualization
- A folder named "Cell04_ll" will be genreated with a file named "particles.csv", which can be loaded in Vutara SRX Software (Bruker) for 3D visualization and rendering
For any questions / comments about this software, please contact Bewersdorf Lab.
Copyright (c) 2020 Bewersdorf Lab, Yale Univeristy School of Medcine, USA.
The package is licenced under the GNU GPL.