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Deep Learning Inferred Multiplex ImmunoFluorescence for IHC Image Quantification (https://deepliif.org) [Nature Machine Intelligence'22, CVPR'22, MICCAI'23, Histopathology'23, MICCAI'24]

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Deep-Learning Inferred Multiplex Immunofluorescence for Immunohistochemical Image Quantification

Nature MI'22 | CVPR'22 | MICCAI'23 | Histopathology'23 | MICCAI'24 | Cloud Deployment | Documentation | Support

Reporting biomarkers assessed by routine immunohistochemical (IHC) staining of tissue is broadly used in diagnostic pathology laboratories for patient care. To date, clinical reporting is predominantly qualitative or semi-quantitative. By creating a multitask deep learning framework referred to as DeepLIIF, we present a single-step solution to stain deconvolution/separation, cell segmentation, and quantitative single-cell IHC scoring. Leveraging a unique de novo dataset of co-registered IHC and multiplex immunofluorescence (mpIF) staining of the same slides, we segment and translate low-cost and prevalent IHC slides to more expensive-yet-informative mpIF images, while simultaneously providing the essential ground truth for the superimposed brightfield IHC channels. Moreover, a new nuclear-envelop stain, LAP2beta, with high (>95%) cell coverage is introduced to improve cell delineation/segmentation and protein expression quantification on IHC slides. By simultaneously translating input IHC images to clean/separated mpIF channels and performing cell segmentation/classification, we show that our model trained on clean IHC Ki67 data can generalize to more noisy and artifact-ridden images as well as other nuclear and non-nuclear markers such as CD3, CD8, BCL2, BCL6, MYC, MUM1, CD10, and TP53. We thoroughly evaluate our method on publicly available benchmark datasets as well as against pathologists' semi-quantitative scoring. Trained on IHC, DeepLIIF generalizes well to H&E images for out-of-the-box nuclear segmentation.

DeepLIIF is deployed as a free publicly available cloud-native platform (https://deepliif.org) with Bioformats (more than 150 input formats supported) and MLOps pipeline. We also release DeepLIIF implementations for single/multi-GPU training, Torchserve/Dask+Torchscript deployment, and auto-scaling via Pulumi (1000s of concurrent connections supported); details can be found in our documentation. DeepLIIF can be run locally (GPU required) by pip installing the package and using the deepliif CLI command. DeepLIIF can be used remotely (no GPU required) through the https://deepliif.org website, calling the cloud API via Python, or via the ImageJ/Fiji plugin; details for the free cloud-native platform can be found in our CVPR'22 paper.

© This code is made available for non-commercial academic purposes.

Version Total Downloads

overview_imageOverview of DeepLIIF pipeline and sample input IHCs (different brown/DAB markers -- BCL2, BCL6, CD10, CD3/CD8, Ki67) with corresponding DeepLIIF-generated hematoxylin/mpIF modalities and classified (positive (red) and negative (blue) cell) segmentation masks. (a) Overview of DeepLIIF. Given an IHC input, our multitask deep learning framework simultaneously infers corresponding Hematoxylin channel, mpIF DAPI, mpIF protein expression (Ki67, CD3, CD8, etc.), and the positive/negative protein cell segmentation, baking explainability and interpretability into the model itself rather than relying on coarse activation/attention maps. In the segmentation mask, the red cells denote cells with positive protein expression (brown/DAB cells in the input IHC), whereas blue cells represent negative cells (blue cells in the input IHC). (b) Example DeepLIIF-generated hematoxylin/mpIF modalities and segmentation masks for different IHC markers. DeepLIIF, trained on clean IHC Ki67 nuclear marker images, can generalize to noisier as well as other IHC nuclear/cytoplasmic marker images.

Prerequisites

  1. Python 3.8
  2. Docker

Installing deepliif

DeepLIIF can be pip installed:

$ conda create --name deepliif_env python=3.8
$ conda activate deepliif_env
(deepliif_env) $ conda install -c conda-forge openjdk
(deepliif_env) $ pip install deepliif

The package is composed of two parts:

  1. A library that implements the core functions used to train and test DeepLIIF models.
  2. A CLI to run common batch operations including training, batch testing and Torchscipt models serialization.

You can list all available commands:

(venv) $ deepliif --help
Usage: deepliif [OPTIONS] COMMAND [ARGS]...

Options:
  --help  Show this message and exit.

Commands:
  prepare-testing-data   Preparing data for testing
  serialize              Serialize DeepLIIF models using Torchscript
  test                   Test trained models
  train                  General-purpose training script for multi-task...

Note: You might need to install a version of PyTorch that is compatible with your CUDA version. Otherwise, only the CPU will be used. Visit the PyTorch website for details. You can confirm if your installation will run on the GPU by checking if the following returns True:

import torch
torch.cuda.is_available()

Training Dataset

For training, all image sets must be 512x512 and combined together in 3072x512 images (six images of size 512x512 stitched together horizontally). The data need to be arranged in the following order:

XXX_Dataset 
    ├── train
    └── val

We have provided a simple function in the CLI for preparing data for training.

  • To prepare data for training, you need to have the image dataset for each image (including IHC, Hematoxylin Channel, mpIF DAPI, mpIF Lap2, mpIF marker, and segmentation mask) in the input directory. Each of the six images for a single image set must have the same naming format, with only the name of the label for the type of image differing between them. The label names must be, respectively: IHC, Hematoxylin, DAPI, Lap2, Marker, Seg. The command takes the address of the directory containing image set data and the address of the output dataset directory. It first creates the train and validation directories inside the given output dataset directory. It then reads all of the images in the input directory and saves the combined image in the train or validation directory, based on the given validation_ratio.
deepliif prepare-training-data --input-dir /path/to/input/images
                               --output-dir /path/to/output/images
                               --validation-ratio 0.2

Training

To train a model:

deepliif train --dataroot /path/to/input/images 
               --name Model_Name 

or

python train.py --dataroot /path/to/input/images 
                --name Model_Name 
  • To view training losses and results, open the URL http://localhost:8097. For cloud servers replace localhost with your IP.
  • Epoch-wise intermediate training results are in DeepLIIF/checkpoints/Model_Name/web/index.html.
  • Trained models will be by default be saved in DeepLIIF/checkpoints/Model_Name.
  • Training datasets can be downloaded here.

DP: To train a model you can use DP. DP is single-process. It means that all the GPUs you want to use must be on the same machine so that they can be included in the same process - you cannot distribute the training across multiple GPU machines, unless you write your own code to handle inter-node (node = machine) communication. To split and manage the workload for multiple GPUs within the same process, DP uses multi-threading. You can find more information on DP here.

To train a model with DP (Example with 2 GPUs (on 1 machine)):

deepliif train --dataroot <data_dir> --batch-size 6 --gpu-ids 0 --gpu-ids 1

Note that batch-size is defined per process. Since DP is a single-process method, the batch-size you set is the effective batch size.

DDP: To train a model you can use DDP. DDP usually spawns multiple processes. DeepLIIF's code follows the PyTorch recommendation to spawn 1 process per GPU (doc). If you want to assign multiple GPUs to each process, you will need to make modifications to DeepLIIF's code (see doc). Despite all the benefits of DDP, one drawback is the extra GPU memory needed for dedicated CUDA buffer for communication. See a short discussion here. In the context of DeepLIIF, this means that there might be situations where you could use a bigger batch size with DP as compared to DDP, which may actually train faster than using DDP with a smaller batch size. You can find more information on DDP here.

To launch training using DDP on a local machine, use deepliif trainlaunch. Example with 2 GPUs (on 1 machine):

deepliif trainlaunch --dataroot <data_dir> --batch-size 3 --gpu-ids 0 --gpu-ids 1 --use-torchrun "--nproc_per_node 2"

Note that

  1. batch-size is defined per process. Since DDP is a single-process method, the batch-size you set is the batch size for each process, and the effective batch size will be batch-size multiplied by the number of processes you started. In the above example, it will be 3 * 2 = 6.
  2. You still need to provide all GPU ids to use to the training command. Internally, in each process DeepLIIF picks the device using gpu_ids[local_rank]. If you provide --gpu-ids 2 --gpu-ids 3, the process with local rank 0 will use gpu id 2 and that with local rank 1 will use gpu id 3.
  3. -t 3 --log_dir <log_dir> is not required, but is a useful setting in torchrun that saves the log from each process to your target log directory. For example:
deepliif trainlaunch --dataroot <data_dir> --batch-size 3 --gpu-ids 0 --gpu-ids 1 --use-torchrun "-t 3 --log_dir <log_dir> --nproc_per_node 2"
  1. If your PyTorch is older than 1.10, DeepLIIF calls torch.distributed.launch in the backend. Otherwise, DeepLIIF calls torchrun.

Serialize Model

The installed deepliif uses Dask to perform inference on the input IHC images. Before running the test command, the model files must be serialized using Torchscript. To serialize the model files:

deepliif serialize --model-dir /path/to/input/model/files
                   --output-dir /path/to/output/model/files
  • By default, the model files are expected to be located in DeepLIIF/model-server/DeepLIIF_Latest_Model.
  • By default, the serialized files will be saved to the same directory as the input model files.

Testing

To test the model:

deepliif test --input-dir /path/to/input/images
              --output-dir /path/to/output/images
              --model-dir /path/to/the/serialized/model
              --tile-size 512

or

python test.py --dataroot /path/to/input/images
               --results_dir /path/to/output/images
               --checkpoints_dir /path/to/model/files
               --name Model_Name
  • The latest version of the pretrained models can be downloaded here.
  • Before running test on images, the model files must be serialized as described above.
  • The serialized model files are expected to be located in DeepLIIF/model-server/DeepLIIF_Latest_Model.
  • The test results will be saved to the specified output directory, which defaults to the input directory.
  • The tile size must be specified and is used to split the image into tiles for processing. The tile size is based on the resolution (scan magnification) of the input image, and the recommended values are a tile size of 512 for 40x images, 256 for 20x, and 128 for 10x. Note that the smaller the tile size, the longer inference will take.
  • Testing datasets can be downloaded here.

Whole Slide Image (WSI) Inference:
For translation and segmentation of whole slide images, you can simply use the same test command giving path to the directory containing your whole slide images as the input-dir. DeepLIIF automatically reads the WSI region by region, and translate and segment each region separately and stitches the regions to create the translation and segmentation for whole slide image, then saves all masks in the format of ome.tiff in the given output-dir. Based on the available GPU resources, the region-size can be changed.

deepliif test --input-dir /path/to/input/images 
              --output-dir /path/to/output/images 
              --model-dir /path/to/the/serialized/model
              --tile-size 512
              --region-size 20000

If you prefer, it is possible to run the models using Torchserve. Please see the documentation on how to deploy the model with Torchserve and for an example of how to run the inference.

Docker

We provide a Dockerfile that can be used to run the DeepLIIF models inside a container. First, you need to install the Docker Engine. After installing the Docker, you need to follow these steps:

  • Download the pretrained model here and place them in DeepLIIF/model-server/DeepLIIF_Latest_Model.
  • To create a docker image from the docker file:
docker build -t cuda/deepliif .

The image is then used as a base. You can copy and use it to run an application. The application needs an isolated environment in which to run, referred to as a container.

  • To create and run a container:
 docker run -it -v `pwd`:`pwd` -w `pwd` cuda/deepliif deepliif test --input-dir Sample_Large_Tissues --tile-size 512

When you run a container from the image, the deepliif CLI will be available. You can easily run any CLI command in the activated environment and copy the results from the docker container to the host.

ImageJ Plugin

If you don't have access to GPU or appropriate hardware and just want to use ImageJ to run inference, we have also created an ImageJ plugin for your convenience.

DeepLIIF ImageJ Demo

The plugin also supports submitting multiple ROIs at once:

DeepLIIF ImageJ ROI Demo

Cloud Deployment

If you don't have access to GPU or appropriate hardware and don't want to install ImageJ, we have also created a cloud-native DeepLIIF deployment with a user-friendly interface to upload images, visualize, interact, and download the final results.

DeepLIIF Website Demo

Our deployment at deepliif.org also provides virtual slide digitization to generate a single stitched image from a 10x video acquired with a microscope and camera. The video should be captured with the following guidelines to achieve the best results:

  • Brief but complete pauses at every section of the sample to avoid motion artifacts.
  • Significant overlap between pauses so that there is sufficient context for stitching frames together.
  • Methodical and consistent movement over the sample. For example, start at the top left corner, then go all the way to the right, then down one step, then all the way to the left, down one step, etc., until the end of the sample is reached. Again, brief overlapping pauses throughout will allow the best quality images to be generated.

DeepLIIF Website Demo

Cloud API Endpoints

For small images, DeepLIIF can also be accessed programmatically through an endpoint by posting a multipart-encoded request containing the original image file, along with optional parameters including postprocessing thresholds:

POST /api/infer

File Parameter:

  img (required)
    Image on which to run DeepLIIF.

Query String Parameters:

  resolution
    Resolution used to scan the slide (10x, 20x, 40x). Default is 40x.

  pil
    If present, use Pillow to load the image instead of Bio-Formats. Pillow is
    faster, but works only on common image types (png, jpeg, etc.).

  slim
    If present, return only the refined segmentation result image.

  nopost
    If present, do not perform postprocessing (returns only inferred images).

  prob_thresh
    Probability threshold used in postprocessing the inferred segmentation map
    image. The segmentation map value must be above this value in order for a
    pixel to be included in the final cell segmentation. Valid values are an
    integer in the range 0-254. Default is 150.

  size_thresh
    Lower threshold for size gating the cells in postprocessing. Segmented
    cells must have more pixels than this value in order to be included in the
    final cell segmentation. Valid values are 0, a positive integer, or 'auto'.
    'Auto' will try to automatically determine this lower bound for size gating
    based on the distribution of detected cell sizes. Default is 'auto'.

  size_thresh_upper
    Upper threshold for size gating the cells in postprocessing.  Segmented
    cells must have less pixels that this value in order to be included in the
    final cell segmentation. Valid values are a positive integer or 'none'.
    'None' will use no upper threshold in size gating. Default is 'none'.

  marker_thresh
    Threshold for the effect that the inferred marker image will have on the
    postprocessing classification of cells as positive.  If any corresponding
    pixel in the marker image for a cell is above this threshold, the cell will
    be classified as being positive regardless of the values from the inferred
    segmentation image. Valid values are an integer in the range 0-255, 'none',
    or 'auto'. 'None' will not use the marker image during classification.
    'Auto' will automatically determine a threshold from the marker image.
    Default is 'auto'.

For example, in Python:

import os
import json
import base64
from io import BytesIO

import requests
from PIL import Image

# Use the sample images from the main DeepLIIF repo
images_dir = './Sample_Large_Tissues'
filename = 'ROI_1.png'

root = os.path.splitext(filename)[0]

res = requests.post(
    url='https://deepliif.org/api/infer',
    files={
        'img': open(f'{images_dir}/{filename}', 'rb'),
    },
    params={
        'resolution': '40x',
    },
)

data = res.json()

def b64_to_pil(b):
    return Image.open(BytesIO(base64.b64decode(b.encode())))

for name, img in data['images'].items():
    with open(f'{images_dir}/{root}_{name}.png', 'wb') as f:
        b64_to_pil(img).save(f, format='PNG')

with open(f'{images_dir}/{root}_scoring.json', 'w') as f:
    json.dump(data['scoring'], f, indent=2)
print(json.dumps(data['scoring'], indent=2))

Note that since this is a single request to send the image and receive the results, processing must complete within the timeout period (typically about one minute). If your request is receiving a 504 status code, please try a smaller image or install the deepliif package as detailed above to run the process locally.

If you have previously run DeepLIIF on an image and want to postprocess it with different thresholds, the postprocessing routine can be called directly using the previously inferred results:

POST /api/postprocess

File Parameters:

  img (required)
    Image on which DeepLIIF was run.

  seg_img (required)
    Inferred segmentation image previously generated by DeepLIIF.

  marker_img (optional)
    Inferred marker image previously generated by DeepLIIF.  If this is
    omitted, then the marker image will not be used in classification.

Query String Parameters:

  resolution
    Resolution used to scan the slide (10x, 20x, 40x). Default is 40x.

  pil
    If present, use Pillow to load the original image instead of Bio-Formats.
    Pillow is faster, but works only on common image types (png, jpeg, etc.).
    Pillow is always used to open the seg_img and marker_img files.

  prob_thresh
    Probability threshold used in postprocessing the inferred segmentation map
    image. The segmentation map value must be above this value in order for a
    pixel to be included in the final cell segmentation. Valid values are an
    integer in the range 0-254. Default is 150.

  size_thresh
    Lower threshold for size gating the cells in postprocessing. Segmented
    cells must have more pixels than this value in order to be included in the
    final cell segmentation. Valid values are 0, a positive integer, or 'auto'.
    'Auto' will try to automatically determine this lower bound for size gating
    based on the distribution of detected cell sizes. Default is 'auto'.

  size_thresh_upper
    Upper threshold for size gating the cells in postprocessing.  Segmented
    cells must have less pixels that this value in order to be included in the
    final cell segmentation. Valid values are a positive integer or 'none'.
    'None' will use no upper threshold in size gating. Default is 'none'.

  marker_thresh
    Threshold for the effect that the inferred marker image will have on the
    postprocessing classification of cells as positive.  If any corresponding
    pixel in the marker image for a cell is above this threshold, the cell will
    be classified as being positive regardless of the values from the inferred
    segmentation image. Valid values are an integer in the range 0-255, 'none',
    or 'auto'. 'None' will not use the marker image during classification.
    'Auto' will automatically determine a threshold from the marker image.
    Default is 'auto'. (If marker_img is not supplied, this has no effect.)

For example, in Python:

import os
import json
import base64
from io import BytesIO

import requests
from PIL import Image

# Use the sample images from the main DeepLIIF repo
images_dir = './Sample_Large_Tissues'
filename = 'ROI_1.png'

root = os.path.splitext(filename)[0]

res = requests.post(
    url='https://deepliif.org/api/infer',
    files={
        'img': open(f'{images_dir}/{filename}', 'rb'),
        'seg_img': open(f'{images_dir}/{root}_Seg.png', 'rb'),
        'marker_img': open(f'{images_dir}/{root}_Marker.png', 'rb'),
    },
    params={
        'resolution': '40x',
        'pil': True,
        'size_thresh': 250,
    },
)

data = res.json()

def b64_to_pil(b):
    return Image.open(BytesIO(base64.b64decode(b.encode())))

for name, img in data['images'].items():
    with open(f'{images_dir}/{root}_{name}.png', 'wb') as f:
        b64_to_pil(img).save(f, format='PNG')

with open(f'{images_dir}/{root}_scoring.json', 'w') as f:
    json.dump(data['scoring'], f, indent=2)
print(json.dumps(data['scoring'], indent=2))

Synthetic Data Generation

The first version of DeepLIIF model suffered from its inability to separate IHC positive cells in some large clusters, resulting from the absence of clustered positive cells in our training data. To infuse more information about the clustered positive cells into our model, we present a novel approach for the synthetic generation of IHC images using co-registered data. We design a GAN-based model that receives the Hematoxylin channel, the mpIF DAPI image, and the segmentation mask and generates the corresponding IHC image. The model converts the Hematoxylin channel to gray-scale to infer more helpful information such as the texture and discard unnecessary information such as color. The Hematoxylin image guides the network to synthesize the background of the IHC image by preserving the shape and texture of the cells and artifacts in the background. The DAPI image assists the network in identifying the location, shape, and texture of the cells to better isolate the cells from the background. The segmentation mask helps the network specify the color of cells based on the type of the cell (positive cell: a brown hue, negative: a blue hue).

In the next step, we generate synthetic IHC images with more clustered positive cells. To do so, we change the segmentation mask by choosing a percentage of random negative cells in the segmentation mask (called as Neg-to-Pos) and converting them into positive cells. Some samples of the synthesized IHC images along with the original IHC image are shown below.

IHC_Gen_imageOverview of synthetic IHC image generation. (a) A training sample of the IHC-generator model. (b) Some samples of synthesized IHC images using the trained IHC-Generator model. The Neg-to-Pos shows the percentage of the negative cells in the segmentation mask converted to positive cells.

We created a new dataset using the original IHC images and synthetic IHC images. We synthesize each image in the dataset two times by setting the Neg-to-Pos parameter to %50 and %70. We re-trained our network with the new dataset. You can find the new trained model here.

Registration

To register the de novo stained mpIF and IHC images, you can use the registration framework in the 'Registration' directory. Please refer to the README file provided in the same directory for more details.

Contributing Training Data

To train DeepLIIF, we used a dataset of lung and bladder tissues containing IHC, hematoxylin, mpIF DAPI, mpIF Lap2, and mpIF Ki67 of the same tissue scanned using ZEISS Axioscan. These images were scaled and co-registered with the fixed IHC images using affine transformations, resulting in 1264 co-registered sets of IHC and corresponding multiplex images of size 512x512. We randomly selected 575 sets for training, 91 sets for validation, and 598 sets for testing the model. We also randomly selected and manually segmented 41 images of size 640x640 from recently released BCDataset which contains Ki67 stained sections of breast carcinoma with Ki67+ and Ki67- cell centroid annotations (for cell detection rather than cell instance segmentation task). We split these tiles into 164 images of size 512x512; the test set varies widely in the density of tumor cells and the Ki67 index. You can find this dataset here.

We are also creating a self-configurable version of DeepLIIF which will take as input any co-registered H&E/IHC and multiplex images and produce the optimal output. If you are generating or have generated H&E/IHC and multiplex staining for the same slide (de novo staining) and would like to contribute that data for DeepLIIF, we can perform co-registration, whole-cell multiplex segmentation via ImPartial, train the DeepLIIF model and release back to the community with full credit to the contributors.

Support

Please use the GitHub Issues tab for discussion, questions, or to report bugs related to DeepLIIF.

License

© Nadeem Lab - DeepLIIF code is distributed under Apache 2.0 with Commons Clause license, and is available for non-commercial academic purposes.

Acknowledgments

This code is inspired by CycleGAN and pix2pix in PyTorch.

Reference

If you find our work useful in your research or if you use parts of this code or our released dataset, please cite the following papers:

@article{ghahremani2022deep,
  title={Deep learning-inferred multiplex immunofluorescence for immunohistochemical image quantification},
  author={Ghahremani, Parmida and Li, Yanyun and Kaufman, Arie and Vanguri, Rami and Greenwald, Noah and Angelo, Michael and Hollmann, Travis J and Nadeem, Saad},
  journal={Nature Machine Intelligence},
  volume={4},
  number={4},
  pages={401--412},
  year={2022},
  publisher={Nature Publishing Group}
}

@article{ghahremani2022deepliifui,
  title={DeepLIIF: An Online Platform for Quantification of Clinical Pathology Slides},
  author={Ghahremani, Parmida and Marino, Joseph and Dodds, Ricardo and Nadeem, Saad},
  journal={Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition (CVPR)},
  pages={21399--21405},
  year={2022}
}

@article{ghahremani2023deepliifdataset,
  title={An AI-Ready Multiplex Staining Dataset for Reproducible and Accurate Characterization of Tumor Immune Microenvironment},
  author={Ghahremani, Parmida and Marino, Joseph and Hernandez-Prera, Juan and V. de la Iglesia, Janis and JC Slebos, Robbert and H. Chung, Christine and Nadeem, Saad},
  journal={International Conference on Medical Image Computing and Computer-Assisted Intervention (MICCAI)},
  volume={14225},
  pages={704--713},
  year={2023}
}

@article{nadeem2023ki67validationMTC,
  author = {Nadeem, Saad and Hanna, Matthew G and Viswanathan, Kartik and Marino, Joseph and Ahadi, Mahsa and Alzumaili, Bayan and Bani, Mohamed-Amine and Chiarucci, Federico and Chou, Angela and De Leo, Antonio and Fuchs, Talia L and Lubin, Daniel J and Luxford, Catherine and Magliocca, Kelly and Martinez, Germán and Shi, Qiuying and Sidhu, Stan and Al Ghuzlan, Abir and Gill, Anthony J and Tallini, Giovanni and Ghossein, Ronald and Xu, Bin},
  title = {Ki67 proliferation index in medullary thyroid carcinoma: a comparative study of multiple counting methods and validation of image analysis and deep learning platforms},
  journal = {Histopathology},
  volume = {83},
  number = {6},
  pages = {981--988},
  year = {2023},
  doi = {https://doi.org/10.1111/his.15048}
}

@article{zehra2024deepliifstitch,
  author = {Zehra, Talat and Marino, Joseph and Wang, Wendy and Frantsuzov, Grigoriy and Nadeem, Saad},
  title = {Rethinking Histology Slide Digitization Workflows for Low-Resource Settings},
  journal = {International Conference on Medical Image Computing and Computer-Assisted Intervention (MICCAI)},
  volume = {15004},
  pages = {427--436},
  year = {2024}
}