Mitochondria Segmentation


Mitochondria are the primary energy providers for cell activities, thus essential for metabolism. Quantification of the size and geometry of mitochondria is not only crucial to basic neuroscience research, but also informative to clinical studies including, but not limited to, bipolar disorder and diabetes.

This tutorial has two parts. In the first part, you will learn how to make pixel-wise class prediction on the widely used benchmark dataset released by Lucchi et al. in 2012. In the second part, you will learn how to predict the instance masks of individual mitochondrion from the large-scale MitoEM dataset released by Wei et al. in 2020.

Semantic Segmentation

This section provides step-by-step guidance for mitochondria segmentation with the EM benchmark datasets released by Lucchi et al. (2012). We consider the task as a semantic segmentation task and predict the mitochondria pixels with encoder-decoder ConvNets similar to the models used in affinity prediction in neuron segmentation. The evaluation of the mitochondria segmentation results is based on the F1 score and Intersection over Union (IoU).


Different from other EM connectomics datasets used in the tutorials, the dataset released by Lucchi et al. is an isotropic dataset, which means the spatial resolution along all three axes is the same. Therefore a completely 3D U-Net and data augmentation along z-x and z-y planes besides x-y planes are preferred.

All the scripts needed for this tutorial can be found at pytorch_connectomics/scripts/. Need to pass the argument --config-file configs/Lucchi-Mitochondria.yaml during training and inference to load the required configurations for this task. The pytorch dataset class of lucchi data is


Qualitative results of the model prediction on the mitochondria segmentation dataset released by Lucchi et al., without any post-processing.

  1. Get the dataset:

    Download the dataset from our server:


    For description of the data please check the author page.

  2. Run the training script:

    $ source activate py3_torch
    $ CUDA_VISIBLE_DEVICES=0,1,2,3,4,5,6,7 python -u scripts/ \
      --config-file configs/Lucchi-Mitochondria.yaml
  3. Visualize the training progress:

    $ tensorboard --logdir runs
  4. Run inference on test image volume:

    $ source activate py3_torch
    $ CUDA_VISIBLE_DEVICES=0,1,2,3,4,5,6,7 python -u scripts/ \
      --config-file configs/Lucchi-Mitochondria.yaml --inference \
      --checkpoint outputs/Lucchi_mito_baseline/volume_100000.pth.tar
  5. Since the ground-truth label of the test set is public, we can run the evaluation locally:

    from connectomics.utils.evaluation import get_binary_jaccard
    pred = pred / 255. # output is casted to uint8 with range [0,255].
    gt = (gt!==0).astype(np.uint8)
    thres = [0.4, 0.6, 0.8] # evaluate at multiple thresholds.
    scores = get_binary_jaccard(pred, gt, thres)

    The prediction can be further improved by conducting median filtering to remove noise:

    from connectomics.utils.evaluation import get_binary_jaccard
    from connectomics.utils.processing import binarize_and_median
    pred = pred / 255. # output is casted to uint8 with range [0,255].
    pred = binarize_and_median(pred, size=(7,7,7), thres=0.8)
    gt = (gt!==0).astype(np.uint8)
    scores = get_binary_jaccard(pred, gt) # prediction is already binarized

Our pretained model achieves a foreground IoU and IoU of 0.892 and 0.943 on the test set, respectively. The results are better or on par with state-of-the-art approaches. Please check for detailed performance comparison and the pre-trained models.

Instance Segmentation

This section provides step-by-step guidance for mitochondria segmentation with our benchmark datasets MitoEM. We consider the task as 3D instance segmentation task and provide three different confiurations of the model output. The model is unet_res_3d, similar to the one used in neuron segmentation. The evaluation of the segmentation results is based on the AP-75 (average precision with an IoU threshold of 0.75).


Complex mitochondria in the MitoEM dataset:(a) mitochondria-on-a-string (MOAS), and (b) dense tangle of touching instances. Those challenging cases are prevalent but not covered in previous datasets.


The MitoEM dataset has two sub-datasets MitoEM-Rat and MitoEM-Human based on the source of the tissues. Three training configuration files on MitoEM-Rat are provided in pytorch_connectomics/configs/MitoEM/ for different learning setting as described in this paper.


Since the dataset is very large and can not be directly loaded into memory, we use the dataset class that only loads part of the whole volume by opening involved PNG or TIFF images.

  1. Introduction to the dataset:

    On the Harvard RC cluster, the datasets can be found at:




    For the public link of the dataset, check the project page.

    Dataset description:

    • im: includes 1,000 single-channel *.png files (4096x4096) of raw EM images (with a spatial resolution of 30x8x8 nm). The 1,000 images are splited into 400, 100 and 500 slices for training, validation and inference, respectively.

    • mito: includes 500 single-channel *.png files (4096x4096) of instance labels. The files are splited into 400 and 100 slices for training and validation. The ground-truth annotation of the test set (rest 500 slices) is not publicly provided but can be evaluated online at the MitoEM challenge page.

    • *.json: dictionary contains paths to the *.png files and metadata of the datasets.

  2. Configure *.yaml files for different learning targets:

    • MitoEM-R-A.yaml: output 3 channels for predicting the affinty between voxels.

    • MitoEM-R-AC.yaml: output 4 channels for predicting both affinity and instance contour.

    • MitoEM-R-BC.yaml: output 2 channels for predicting both the binary foreground mask and instance contour. This configuration achieves the best overall performance according to our experiments.

  3. Run training script for the U3D-BC model:


    By default the path of images and labels are not specified. To run the training scripts, please revise the DATASET.IMAGE_NAME, DATASET.LABEL_NAME, DATASET.OUTPUT_PATH and DATASET.INPUT_PATH options in configs/MitoEM-R-*.yaml. The options can also be given as command-line arguments without changing of the yaml configuration files.

    $ source activate py3_torch
    $ python -u scripts/ --config-file configs/MitoEM-R-BC.yaml
  4. Visualize the training progress. More info here:

    $ tensorboard --logdir outputs/MitoEM_R_BC/
  5. Run inference on validation/test image volumes (suppose the model is optimized for 100k iterations):

    $ source activate py3_torch
    $ python -u scripts/ \
      --config-file configs/MitoEM-R-BC.yaml --inference \
      --checkpoint outputs/MitoEM_R_BC/checkpoint_100000.pth.tar


    Please change the INFERENCE.IMAGE_NAME INFERENCE.OUTPUT_PATH INFERENCE.OUTPUT_NAME options in configs/MitoEM-R-*.yaml.

  6. Merge output volumes and run watershed segmentation:

    As mentioned before, the dataset is very large and can hardly be directly loaded into memory for processing. Therefore our code run prediction on smaller chunks sequentially, which produces multiple *.h5 files with the coordinate information. To merge the chunks into a single volume and apply the segmentation algorithm:

    import glob
    import numpy as np
    from import readvol
    from connectomics.utils.processing import bc_watershed
    output_files = 'outputs/MitoEM_R_BC/test/*.h5' # output folder with chunks
    chunks = glob.glob(output_files)
    vol_shape = (2, 500, 4096, 4096) # MitoEM test set
    pred = np.ones(vol_shape, dtype=np.uint8)
    for x in chunks:
        pos = x.strip().split("/")[-1]
        print("process chunk: ", pos)
        pos = pos.split("_")[-1].split(".")[0].split("-")
        pos = list(map(int, pos))
        chunk = readvol(x)
        pred[:, pos[0]:pos[1], pos[2]:pos[3], pos[4]:pos[5]] = chunk
    # This function process the array in numpy.float64 format.
    # Please allocate enough memory for processing.
    segm = bc_watershed(pred, thres1=0.85, thres2=0.6, thres3=0.8, thres_small=1024)

    Then the segmentation map should be ready to be submitted to the MitoEM challenge website for evaluation. Please note that this tutorial only take the MitoEM-Rat set as an example. The MitoEM-Human set also need to be segmented for online evaluation.