A set of tools to simulate expansion microscopy experiments under different labeling and imaging conditions.
The software is fully written in Python. The following section covers basic installation and usage of SimExm.
The code works in python2 exclusively. It should be easy to convert it to python3 but it's untested. I strongly suggest creating a virtual environment, with virtualenv or anaconda and then use the pip command above to install all the dependencies inside the environment. For instance, you can create a new environment "sim" by running:
conda create -n sim python=2.7
Which will modify the PYTHONPATH so that all futute programs installed through pip end up in the same virtual environment. To start your new environment and activate the change of path run:
source activate sim
All dependencies currently used are: numpy, Pillow, scipy, images2gif, configobj and tifffile. To install all at once, from the terminal, do:
pip install -r requirements.txt
or if you didn't use conda or a virtual environment:
sudo pip install -r requirements.txt
Finally, you can go ahead and compile the psf module, courtesy of Christoph Gohlke: http://www.lfd.uci.edu/~gohlke/.
python setup.py build_ext --inplace
Once the module has successfully compiled, you are ready to go!
The simulation is used through configuration files. We use the configobj module which provides a nice syntax and validation of the config files. The specifications are outlines in the configspecs.ini file. Example configurations can be found under ./examples. To create a new configuration file copy one of the templates in ./examples, and modify it to fit your needs. The config parameters are explained below. For more information see the in-code documentation.
| Parameter | Type | Range | Description |
|---|---|---|---|
| image_path | string | - | indicates the location of the ground truth data. The data should be in the form of an image sequence, where each image represents a slice of the ground truth volume or as Tiff stack. In each image, pixel values should indicate what cell the pixel belongs to, or 0 if the pixel is located in extra-cellular space. This is true for both formats, file names will be ingored. |
| offset | z, x, y tuple | - | tuple of length 3 representing where to start loading the data, in pixels. For instance, an offset of (10, 50, 50) means that images are loaded starting the 10th image in the given directory and each imaage is cropped so that the top left corner is at (50, 50). |
| bounds | z, x, y tuple) | - | tuple of length 3 representing the size of the data to load. Could be smaller than the actual size of the ground truth. This is the size in number of voxels. |
| format | string | one of "tiff" or "image sequence" | Specifies which format is used in the groudn truth. If tiff, the path to the tiff stack should be specified in "image_path", otherwise a path to the folder containing the image sequence should be given in image_path. |
| gt_cells | string | one of "merged" or "splitted" | If merged, all cells are expected to be loaded from the same volume. If splitted, a different volume is expected for each cell. If the chosen format is tiff stack, the image_path should contain a list of tiff stacks. Otherwise, it should contain a list of folders, each containing the image sequence for a given cell. |
| voxel_dim | z, x, y tuple | - | tuple of length 3 representing the dimensions of a single voxel, in nanometers. |
| isotropic | boolean | True or False | Choose False if the voxel dimension of the ground truth data is not isotropic (i.e z voxel dim is different than xy dim). |
| regions | - | - | a subsection in the configuration file which may contian many different regions. A region has a single parameter, region_path, which is a string pointing to where the data for that region is. By default the software automatically computes the cytosol and membrane regions, but additional annotations may be available. They are loaded by overlapping the region with the orgiinal cell segmentaiton to figure out which part of the cell is in the given region, for each cell. See synapse.ini for an example. |
| Parameter | Type | Range | Description |
|---|---|---|---|
| global_density | float | between 0.0 and 1.0 | the global labeling density, determines the number of cells that can be labeled by any of the fluorophores |
Additionaly, the labeling section should contain a subsection for each fluorophore used. See the example in brianbow_membrane.ini. Each subsection should contain the following parameters:
| Parameter | Type | Range | Description |
|---|---|---|---|
| fluorophore | string | one of "ATTO390", "ATTO425", " ATTO430LS", "ATTO488", "ATTO490LS", "ATTO550", "ATTO647N", "ATTO700", "Alexa350" and "Alexa790" | the fluorophore to use. More info in src/fluors.py |
| region | string | one of "cytosol", "membrane" or any additional regions specified in the ground truth | the region to annotate with the above fluorophore |
| density | float | between 0.0 and 1.0 | the proportion of cells in the volume to annotate with the above fluorophore. |
| protein_density | float | greater than 0.0 | the density of proteins to label the sample with. 1.0 is 1 fluorophore per nm^3, which is not really realistic and should provide a good upperbound. |
| protein_noise | float | between 0.0 and 1.0 | the proportion of proteins that fly away from the labeled region. Uses a gaussian distirbution around the original location. The standard devitation for that gaussian can be modified directly in the noise function in src/labeling.py but the default should provide fairly realistic results. |
| antibody_amp | float | greater than 1.0 | amplifies the labeling, as well as the noise. Tipically 5.0 or 10.0. |
| single_neuron | boolean | True or False | if True, a random cell is chosen and is the only one labeled with the above fluorophore |
| Parameter | Type | Range | Description |
|---|---|---|---|
| factor | float | greater than 1.0 | the expansion factor use in the expansion microscopy protocol. |
| Parameter | Type | Range | Description |
|---|---|---|---|
| type | string | one of 'confocal', 'widefield' or 'two photon' | type of microscope to use |
| numerical_aperture | float | greater than 0.0 | the numerical aperture of the system |
| refractory_index | float | greater than 1.0 | the refractory index of the system |
| focal_plane_depth | integer | greater than 1 | the thickness of a z-slice in nanometers |
| objective_back_aperture | float | greater than 0.0 | the objective back aperture of the system |
| exposure_time | float | greater than 0 | how long photons are detected, in seconds |
| objective_efficiency | float | between 0.0 and 1.0 | the percentage of efficiency of the objective |
| detector_efficiency | float | between 0.0 and 1.0 | the percentage of efficiency of the detector |
| objective_factor | float | greater than 1.0 | the objective lens, tipically 20x, 40x |
| pixel_size | integer | greater than 1 | the size of an output pixel in the microscope, in nanometers |
| pinhole_radius | float | greater than 0.0 | the pinhole radius, in micrometers |
| baseline_noise | integer | greater than 0 | the average number of baseline photons detected by the system |
| channels | - | - | subsection containing a multiple channel parameters for different lasers. Each subsection has the following parameters. See brainbow_membrane.ini for an example on how to use multiple channels. |
| laser_wavelength | integer | between 200 and 1000 | the wavelength of the laser, in nanometers |
| laser_power | float | greater than 1.0 | the power of the laser, in Watts |
| laser_percentage | float | between 0.0 and 1.0 | proportion of the laser power to use |
| laser_filter | min, max integer tuple | - | the minimum and maximum wavelengths of the filter, in nanometers |
The simualtion outputs a simualted stack and the corresponding ground truth, which can be used for validation or training. Output formats include: tiff stacks, gif or png sequence. The simulation stack may be outputted in separate channels or merged into multiple RGB volumes. If the number of channels is greater than 3, an RGB volume may be created for every 3 channels.
The ground truth is stored on a per fluorophore basis. For each fluorophore the output may be spliited into a volume for each cell or grouped into a single volume containing all cells labeled by that fluorophore. The parameters are outlined below.
| Parameter | Type | Range | Description |
|---|---|---|---|
| name | string | - | a name for the experiment |
| path | path | - | where to store the experiement's output |
| format | string | one of 'tiff', 'gif' or 'image sequence' | determines to output format for both the simulated stack and the ground truth |
| sim_channels | string | one of "merged" or "splitted" | if merged, and RGB volume is made for every 3 channels, otherwise a stack is made for each channel |
| gt_cells | string | one of "merged ot "splitted" | if merged, all cells are grouped in the same stack, otherwise, a volume if made for each cell |
| gt_region | string | one of 'membrane', 'cytosol' or any additional annotation specified in the ground truth | the cell region to use in the output, may be different that the annotated regions in the labeling layers |
Once the config.ini file is ready, make sure that you have activated your virtual environment if you have one (using source activate environment_name). Then, run the simulation from the terminal by using the following command:
python run.py path_to_config/config.ini
The script currently takes two options: '-h' which displays the command line help, and '-v' which displays the output of the simulation in a new window.
Note that the simulation may require a decent amount of memory for large volumes. A computer with 4G of RAM can usually handle a volume of about 500 x 500 x 500 voxels without problems. If you run into memory issues, consider using a computer with more RAM.
Provided under BSD license
Copyright (c) 2017, Jeremy Wohlwend
All rights reserved.
Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:
- Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.
- Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.
- Neither the name of SimExm nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission.
THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL JEREMY WOHLWEND BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
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