Imaging Mass Cytometry (IMC) is an emerging multiplexed imaging technology for analyzing complex microenvironments that has the ability to detect the spatial distribution of at least 40 cell markers. However, this new modality has unique image data processing requirements, particularly when applying this technology to patient tissue specimens. In these cases, signal-to-noise ratio (SNR) for particular markers can be low despite optimization of staining conditions, and the presence of pixel intensity artifacts can deteriorate image quality and the performance of downstream analysis. Here we demonstrate a content aware pipeline, IMC-Denoise, to restore IMC images. Specifically, we deploy (i) a Differential Intensity Map-based Restoration (DIMR) algorithm for removing hot pixels and (ii, iii) a self-supervised Deep learning algorithm for Shot Noise image Filtering (DeepSNiF). IMC-Denoise enables adaptive hot pixel removal without loss of resolution and delivers significant SNR improvement to a diverse set of IMC channels and datasets. Here we show how to implement IMC-Denoise and develop the software package IMC_Denoise. We hope this package could help the researchers in the field of mass cytometry imaging.
Denoise lymphocyte antibody-stained images
Denoise other antibody-stained images
- Directory structure of IMC_Denoise
- Customize environment for IMC_Denoise
- Implement IMC_Denoise
- Contact
- References
IMC_Denoise
|---IMC_Denoise
|---|---IMC_Denoise_main
|---|---|---DIMR.py
|---|---|---DeepSNiF.py
|---|---|---DeepSNiF_model.py
|---|---|---loss_functions.py
|---|---DeepSNiF_utils
|---|---|---DeepSNiF_DataGenerator.py
|---|---|---DeepSNiF_TrainGenerator.py
|---|---Anscombe_transform
|---|---|---Anscombe_transform_functions.py
|---|---|---Anscombe_vectors.mat
|---Jupyter_Notebook_examples
|---|---IMC_Denoise_Train_and_Predict.ipynb
|---|---IMC_Denoise_Train.ipynb
|---|---IMC_Denoise_Predict.ipynb
|---scripts
|---|---Data_generation_DeepSNiF_script.py
|---|---Training_DeepSNiF_script.py
|---|---Generate_data_and_training_DeepSNiF_script.py
|---|---Predict_DIMR_script.py
|---|---Predict_IMC_Denoise_script.py
- IMC_Denoise implements DIMR and DeepSNiF algorithms to remove hot pixels and filter shot noise in IMC images, respectively.
- Jupyter Notebooks and scripts include several examples to implement IMC_Denoise algorithms.
- Windows 10 64bit
- Python 3.6
- Tensorflow 2.2.0
- Keras 2.3.1
- NVIDIA GPU + CUDA
- Note that Linux also works with such configurations
- Create a virtual environment and install tensorflow-gpu and keras (we run all the codes in a Anaconda Powershell Prompt).
$ conda create -n 'IMC_Denoise' python=3.6
$ conda activate IMC_Denoise (some systems recommend: source activate IMC_Denoise)
$ conda install -c anaconda brotlipy
$ pip install tensorflow==2.2.0 keras==2.3.1
$ conda install -c anaconda cudnn=7.6.5 cudatoolkit=10.1.243
$ pip install jupyter
- In case above commends do not work, please try:
$ conda create -n IMC_Denoise python=3.6 anaconda
$ conda activate IMC_Denoise (some systems recommend: source activate IMC_Denoise)
$ pip install tensorflow==2.2.0 keras==2.3.1
$ conda install -c anaconda cudnn=7.6.5 cudatoolkit=10.1.243
- Download the source code and install the package in your folder.
$ git clone https://github.com/PENGLU-WashU/IMC_Denoise.git
$ cd IMC_Denoise
$ pip install -e .
- In case the training losses are NaN, please install the following cuda libraries:
conda install -c anaconda cudnn=8.1 cudatoolkit=11.2
- Use dockerfile provided to build reproducible environment with IMC_Denoise.
$ git clone https://github.com/PENGLU-WashU/IMC_Denoise.git
$ cd IMC_Denoise
$ docker build -t imc_denoise:1.0 . --platform linux/amd64
- On LSF cluster to run scripts or jupyter notebooks, respectively:
$ LSF_DOCKER_PORTS="8888:8888" bsub -Is -R 'select[gpuhost,port8888=1]' -gpu "num=1:gmodel=TeslaV100_SXM2_32GB" -a 'docker(imc_denoise:1.0)' /bin/bash
$ cd /IMC_Denoise
$ LSF_DOCKER_PORTS="8888:8888" PATH="/opt/conda/bin:$PATH" bsub -Is -R 'select[gpuhost,port8888=1]' -gpu "num=1:gmodel=TeslaV100_SXM2_32GB" -a 'docker(imc_denoise:latest)' jupyter-notebook --ip=0.0.0.0 --NotebookApp.allow_origin=*
In order to generate a training set for DeepSNiF, the directory structure of raw IMC images must be arranged as follows. Note that the Channel_img names should contain the specific isotope names. For example, "141Pr" in "141Pr-CD38_Pr141.tiff" and "144Nd" in "144Nd-CD14_Nd144.tiff". We define the isotope names as the channel names of the IMC images.
|---Raw_image_directory
|---|---Tissue1_sub_directory
|---|---|---Channel1_img.tiff
|---|---|---Channel2_img.tiff
...
|---|---|---Channel_n_img.tiff
|---|---Tissue2_sub_directory
|---|---|---Channel1_img.tiff
|---|---|---Channel2_img.tiff
...
|---|---|---Channel_n_img.tiff
...
|---|---Tissue_m_sub_directory
|---|---|---Channel1_img.tiff
|---|---|---Channel2_img.tiff
...
|---|---|---Channel_n_img.tiff
IMC_Denoise can now also be run with multi-channel .tiffs, originally intended as a way of smoothly integrating the package with the Steinbock pipeline from the Bodenmiller group (https://github.com/BodenmillerGroup/steinbock).
Multi-channel Directory Structure:
|--- Raw_image_directory
|---|---MCD1_ROI_1.tiff
|---|---MCD1_ROI_2.tiff
|---|---MCD2_ROI_1.tiff
...
...
|---|---MCDn_ROI_n.tiff
This is the structure naturally produced by Steinbock, with the /img directory produced by steinbock being the "Raw_image_directory".
- The DeepSNiF_DataGenerator Class now has an additional attribute: run_type. Set run_type = 'multi_channel_tiff' in the DeepSNiF_DataGenerator() call to allow ingestion of multi-channel tiffs.
- The default behavior remains the same, requiring the single-channel directory structure given above this section. This can be explicitly called with run_type = 'single_channel_tiff'.
- Channel Names with the multi_channel_tiff option MUST BE CALLED AS AN INTEGER, which corresponds to the channel's numbered order in the images, and NOT by isotope name. This is different than the procedure with single channel tiffs. This also means that the order of channels needs to be the same for images to be succesfully processed in one batch, although this should usually be the case for an experiment.
- There is an example jupyter notebook showing an example of how to use the steinbock integration / multi-channel option. Notice in the notebook how the training directory can be set to be a different folder than the directory of the images that you process, or they can be the same. Both, though, need to have the directory structure shown above.
For user-friendly use of the Steinbock Jupyter script we now also provide a CLI version with some options to be directly called from the CLI. Note, that this script is provided for the Steinbock multichannel TIFF Version and currently does not support singlechannel.
| Option | Description | Optional |
|---|---|---|
-t, --train |
Path to the training directory | No |
-i, --input |
Path to the input directory where raw multichannel tiffs are located | No |
-o, --output |
Path to the directory where denoised images will be saved. If not specified, denoised images will overwrite the original images in the input directory. | Yes |
-c, --channels |
Comma-separated list of channels to denoise (e.g., '1,2,3'). If not provided, all channels will be used. | Yes |
-b, --batch_size |
Batch size for training of DeepSNIF, default is 128 | Yes |
-e, --epochs |
Number of Epochs for training, default is 100 | Yes |
-l, --learning_rate |
Initial learning rate, default is 1e-3 | Yes |
-
Please go to https://doi.org/10.5281/zenodo.6533905 and download Raw_IMC_dataset_for_training_supp_table5.zip. Then unzip this file as the folder Raw_IMC_dataset_for_training_supp_table5. This folder contains all the images for DeepSNiF training.
-
We also provide all the images of this human bone marrow IMC dataset, which are compressed in Raw_IMC_dataset_all_supp_table5 and can also be downloaded from https://doi.org/10.5281/zenodo.6533905.
-
Previously generated training sets and trained weights can be accessed from https://doi.org/10.5281/zenodo.7101454. Please refer to our paper for more details.
Now we have added one more hyper-parameter "network_size" (please refer to the hyper-parameter table and the code).
- When setting the parameter as "normal", the original network structure using Resnet and UNet will be applied in training and prediction.
- When setting the parameter as "small", a much smaller network only using UNet will be applied, so that the total parameters decrease from 33,136,320 to 243,488, and the training time decreases by approximately 80%.
- The small network is fit for small datasets (all of our cases work well!). Nevertheless, the normal one can be applied if you have a much larger dataset or the performance of the small one is not ideal.
- We set "small" as the default setting for the parameter "network_size" for all the cases. However, all the trained weights in Zenodo applied "normal" network setting.
- You can even define your own network structure by changing the code in "IMC_Denoise/IMC_Denoise_main/DeepSNiF_model".
| Parameter | Description | Default Value | Data type |
|---|---|---|---|
| n_neighbours | The number of adjacent pixels used to compare with center pixel in DIMR algorithm. | 4 | int |
| n_iter | The iteration number of DIMR algorithm. | 3 | int |
| slide_window_size | The sliding window size in DIMR algorithm. | 3 | int |
| ratio_thresh | The threshold of the sparsity of the generated patch, which is range from 0 to 1. If the percentage of zero-value pixel is larger than this threshold, the corresponding patch will not be saved in the generated training set. | 0.8 | float |
| train_epoches | The training epoches in DeepSNiF. | 200 | int |
| train_batch_size | The training batch size in DeepSNiF. Try smaller value if memory is not enough. | 128 | int |
| lambda_HF | The parameter for Hessian regularization. We recommend to set it as 3e-6. | 3e-6 | float |
| train_initial_lr | Initial training rate. | 1e-3 | float |
| truncated_max_rate | The max_val of the channel is 1.1*(truncated_max_rate*100)-th pixel values, which is used to mitigate the impact of extremely large pixel values. Normally set as 0.99999, 0.9999 or 0.999. | 0.99999 | float |
| val_set_percent | The percentage of patches used as validation set, which ranges from 0 to 1. | 0.15 | float |
| network_size | Decide whether normal (33,136,320 parameters) or small (243,488 parameters) network to be used. | small | str |
- To start Jupyter Notebooks and run the examples.
$ conda activate IMC_Denoise
$ jupyter notebook --notebook-dir=your_folder_of_notebook_examples
- Train and predict the DeepSNiF algorithm separately, in which the generated dataset and trained weights will be saved.
- Train and predict the DeepSNiF algorithm in the same notebook, in which the generated dataset and trained weights will not be saved.
- Activate the IMC_Denoise environment.
$ conda activate IMC_Denoise
- Here we take the images with marker CD38 as an example. For our dataset, CD38 is conjucted with 141Pr. In this case, the "channel_name" should be set as its corresponding isotope name "141Pr".
- Generating training set and train a DeepSNiF model.
- Generate training set of a specific marker channel for DeepSNiF. The generated training data will be saved in a sub-directory "Generated_training_set" of the current folder other than setting a customized folder. For CD38, the saved name will be "training_set_141Pr.npz".
python scripts/Data_generation_DeepSNiF_script.py --channel_name '141Pr' --Raw_directory 'Your_raw_img_directory' --Save_directory 'your_generated_training_set_directory' --n_neighbours '4' --n_iter '3' --slide_window_size '3' --ratio_thresh '0.8'- Train a DeepSNiF network. The generated training set will be loaded from a default folder other than choosing a customized folder. The trained weights will be saved in a sub-directory "trained_weights" of the current folder other than setting a customized folder. Hyper-parameters can be adjusted. Note that when implementing prediction, input the same "trained_weights" name. If your GPU has smaller memory so that it cannot afford a large "train_batch_size" such as 128 or 256, please use a smaller one, e.g. 64, 32.
python scripts/Training_DeepSNiF_script.py --train_set_name 'training_set_141Pr.npz' --train_data_directory 'directory_of_your_training_set' --weights_name 'weights_141Pr-CD38.hdf5' --train_epoches '200' --train_batch_size '128' --val_set_percent '0.15' --lambda_HF '3e-6' --train_initial_lr '1e-3' --truncated_max_rate '0.99999' --network_size 'small'- Generate training set for a specific marker channel and then train a DeepSNiF network. In this process, the generated training set will not be saved in a directory.
python scripts/Generate_data_and_training_DeepSNiF_script.py --channel_name '141Pr' --weights_name 'weights_141Pr-CD38.hdf5' --Raw_directory 'Your_raw_img_directory' --train_epoches '200' --train_batch_size '128' --val_set_percent '0.15' --n_neighbours '4' --n_iter '3' --slide_window_size '3' --ratio_thresh '0.8' --lambda_HF '3e-6' --train_initial_lr '1e-3' --truncated_max_rate '0.99999' --network_size 'small' - Combine multiple generated training sets from different channels into a single training set.
python scripts/Combine_multiple_datasets.py --load_directory 'the_folder_of_the_training_sets_to_be_combined' --save_directory 'the_folder_to_save_the_combined_training_set' --saved_training_set_name" 'the_name_of_the_combined_training_set' - Implement IMC_Denoise to enhance IMC images.
- Implement DIMR for a single IMC image if the SNR of the image is good.
python scripts/Predict_DIMR_script.py --Raw_img_name 'your_raw_img_name(.tiff)' --Denoised_img_name 'your_denoised_img_name(.tiff)' --n_neighbours '4' --n_iter '3' --slide_window_size '3'- Implement DIMR for multiple IMC images if the SNR of the image is good.
python scripts/Predict_DIMR_batch.py --channel_name '141Pr' --load_directory 'raw_image_folders (please refer to Section: Directory structure of IMC_Denoise)' --save_directory 'DIMR_processed_image_folders' --n_neighbours '4' --n_iter '3' --slide_window_size '3'- Implement IMC_Denoise including DIMR and DeepSNiF for a single IMC image if the image is contaminated by hot pixels and suffers from low SNR. The trained weights will be loaded from the default directory other than choosing a customized folder.
python scripts/Predict_IMC_Denoise_script.py --Raw_img_name 'your_raw_img_name(.tiff)' --Denoised_img_name 'your_denoised_img_name(.tiff)' --weights_name 'weights_141Pr-CD38.hdf5' --weights_save_directory 'your_directory_to_save_trained_weights' --n_neighbours '4' --n_iter '3' --slide_window_size '3' --network_size 'small'- Implement IMC_Denoise including DIMR and DeepSNiF for multiple IMC imagse if the images are contaminated by hot pixels and suffers from low SNR. The trained weights will be loaded from the default directory other than choosing a customized folder.
python scripts/Predict_IMC_Denoise_batch.py --channel_name '141Pr' --load_directory 'raw_image_folders (please refer to Section: Directory structure of IMC_Denoise)' --save_directory 'IMC_Denoise_processed_image_folders' --weights_name 'weights_141Pr-CD38.hdf5' --weights_save_directory 'your_directory_to_save_trained_weights' --n_neighbours '4' --n_iter '3' --slide_window_size '3' --network_size 'small' - More specific parameters can also be added and adjusted. Please refer to the scripts files.
Peng Lu - @penglu10 - [email protected]
Project Link: https://github.com/PENGLU-WashU/IMC_Denoise
Lab Website: Thorek Lab WashU
Thanks the contributions of BenCaiello and LukasHats on this project, who help modify the code on Steinbock.
If you find our work useful in your research or if you use parts of this code please consider citing our paper:
Lu P, Oetjen K, Bender D, et al. IMC-Denoise: a content aware pipeline to enhance Imaging Mass Cytometry. Nature Communications, 14(1), 1601, 2023. https://www.nature.com/articles/s41467-023-37123-6
@article{lu2023imc,
title={IMC-Denoise: a content aware denoising pipeline to enhance Imaging Mass Cytometry},
author={Lu, Peng and Oetjen, Karolyn A and Bender, Diane E and Ruzinova, Marianna B and Fisher, Daniel AC and Shim, Kevin G and Pachynski, Russell K and Brennen, W Nathaniel and Oh, Stephen T and Link, Daniel C and others},
journal={Nature Communications},
volume={14},
number={1},
pages={1601},
year={2023},
publisher={Nature Publishing Group UK London}
}
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