Here we describe how to use DECODE locally, i.e., when you want to use it on a regular basis. If you want to test DECODE without installation you can check out the Google Colab notebooks linked on the starting page of this documentation.
Note: This tutorial assumes that you have successfully installed DECODE locally and got your copy of the DECODE Jupyter notebooks. If this is not the case for you, please refer to the installation instructions and follow the step-by-step guide.
A typical workflow for fitting high-density SMLM data with this package is
Bead calibration and extraction of spline coefficients (e.g. in SMAP)
Determine training parameters by a pre-fitting procedure or reasonableguess.
Fitting experimental data
Export, visualization and analysis of fitted data
The first two steps involving SMAP can be skipped and you can start right away with the notebooks in case you want to work with our example data, as we provide the intermediate result files (i.e., the calibration and the training parametrization). If you are working with your own data or want to go through the whole workflow, just start from the beginning. You can find an overview of our data in Data.
Bead calibration with SMAP#
Install the stand-alone version of SMAP from the software section on rieslab.de or if you have MATLAB, download the source-code from GitHub.com/jries/SMAP. On rieslab.de, you can also find the installation instructions and the documentation.
Acquire z-stacks with fluorescent beads (e.g. 100 nm beads). We typically use a z-range of +/- 1000 nm and a step size of 10-50 nm.
In SMAP, use the plugin Analyze / calibrate3DSplinePSF to generate the calibration file. The plugin can be found either via tabs Analyze / sr3D / calibrate3DsplinePSF or menu Plugins / Analyze / sr3D / calibrate3DsplinePSF. More details about the process can be found in the User Guide of SMAP in section 5.4, in the Step by Step Guide for SMAP, and in the original publication Li et al., Nature Methods (2018). Even for two-dimensional data you need a bead calibration, in this case make sure to perform the bidirectional fit.
Determine training parameters with SMAP#
Use the bead calibration to fit your SMLM data. Detailed instructions can be found in the SMAP user guide in section 5, more specifically in section 5.4 for fitting with an experimental PSF.
Use the plugin: Plugins / calibrate / DECODE_training_estimates to estimate the photo-physical parameters of the experiment and to save them into a parameter file. Consult the information of the plugin (accessible via the Info button) for further information.
Training a DECODE model#
The basis for training DECODE is a parametrization of training procedure. This parametrization is described in a simple .yaml file which contains a couple of paths (e.g., the calibration file and your output directory) as well as the parametrization of the simulation which should match the data you want to fit.
In our Training notebook we guide you through the process of creating such a .yaml file that can subsequently be used to start the actual training.
If you have gone through the notebooks already and generated your own param.yaml file, you can skip the following section and go to the regular workflow directly.
First time using DECODE#
To get you up and running, we provide several notebooks that introduce DECODE to you. In total, there are four different notebooks:
Introduction contains a general introduction to DECODE and helps you to get familiar with the framework.
Training guides you through creating a parameter file that you need for training the model. It is based on emitter characteristics determined previously (or provided by the example).
Fitting localizes the single molecules in the high-density data based on the model.
Evaluation gives you an introduction to the post-processing capabilities of DECODE.
To start going through the notebooks, execute the following command in your Terminal/Anaconda Prompt:
conda activate decode_env # if not already activated jupyter lab
This will open up a new browser tab/window. Navigate to the path where you saved the notebooks and go through them. We recommend to start with the Introduction notebook, followed by Training and Fitting.
In practice, you can either write such a .yaml file directly, i.e., by educated guessing your emitter characteristics, or follow the pre-fit routine using SMAP that will auto-generate it.
Once being equipped with your calibration and the parameter file, you can start the training in your Terminal/Anaconda prompt
conda activate decode_env # if not already activated python -m decode.neuralfitter.train.live_engine -p [path to your param].yaml
To monitor the training progress, you can open up a new Terminal window/Anaconda prompt, navigate to the respective folder from before, and start Tensorboard. This is optional and does not have any influence on the training. Note that Tensorboard can be quite slow sometimes.
conda activate decode_env # if not already activated tensorboard --samples_per_plugin images=100 --port=6006 --logdir=runs
Please refer to the Fitting notebook which is described above in First Time using DECODE instructions.
Export from DECODE and import in SMAP for visualization#
DECODE has basic rendering functions but for detailed visualization and analysis you should export your data and load it into SMAP or another SMLM visualization software of your choice.
For loading the data in SMAP, you should save your emitters as an h5 file at the end of the fitting notebook. Under the File tab in SMAP, change the auto loader to Import DECODE .csv/.h5 and Load the exported data. For detailed instructions on post-processing (grouping, filtering, drift correction,…) please consult the SMAP Documentation, more specifically from section 5 onwards in the Getting Started Guide and from section 6 on in the SMAP User Guide. While we recommend using the h5 format in general, for a prototypic input to other software we allow exporting as self-explanatory csv.