Automatic and manual segmentation pipeline¶

[1]:

import brainlit
from brainlit.utils.session import NeuroglancerSession
from brainlit.utils.Neuron_trace import NeuronTrace
from brainlit.algorithms.generate_fragments import adaptive_thresh
import napari
from napari.utils import nbscreenshot
%gui qt5

/opt/buildhome/python3.7/lib/python3.7/site-packages/tqdm/auto.py:22: TqdmWarning: IProgress not found. Please update jupyter and ipywidgets. See https://ipywidgets.readthedocs.io/en/stable/user_install.html
  from .autonotebook import tqdm as notebook_tqdm

Find valid segments¶

In this cell, we set up a NeuroglancerSession object. Since segmentation ID numbers are not in order, we print out a list of valid IDs in some range id_range. Most segment IDs are in range(300), additionally, segments 999 and 1000 are available.

[2]:

"""
# Optional: Print the IDs of segments in Neuroglancer
url = "s3://open-neurodata/brainlit/brain1"
ngl_skel = NeuroglancerSession(url+"_segments", mip=1,use_https=False)
working_ids = []
id_range = 14
for seg_id in range(id_range):
    try:
        segment = ngl_skel.cv.skeleton.get(seg_id)
        working_ids.append(seg_id)
    except:
          pass
print(working_ids)
"""

[2]:

'\n# Optional: Print the IDs of segments in Neuroglancer\nurl = "s3://open-neurodata/brainlit/brain1"\nngl_skel = NeuroglancerSession(url+"_segments", mip=1,use_https=False)\nworking_ids = []\nid_range = 14\nfor seg_id in range(id_range): \n    try:\n        segment = ngl_skel.cv.skeleton.get(seg_id)\n        working_ids.append(seg_id)\n    except:\n          pass\nprint(working_ids)\n'

Download SWC information¶

Download the information contained in a SWC file for labelled vertices of a given seg_id at a valid mip from AWS.

[3]:

"""
seg_id = 13
mip = 2
s3_trace = NeuronTrace(url+"_segments", seg_id, mip)
df = s3_trace.get_df()
df['sample'].size # the number of vertex IDs [1, 2, ..., df['sample'].size]
"""

[3]:

'\nseg_id = 13\nmip = 2\ns3_trace = NeuronTrace(url+"_segments", seg_id, mip)\ndf = s3_trace.get_df()\ndf[\'sample\'].size # the number of vertex IDs [1, 2, ..., df[\'sample\'].size]\n'

[4]:

"""
print(df)
"""

[4]:

'\nprint(df)\n'

Select vertices¶

Select a subset of the vertices in the downloaded SWC to view and segment.

[5]:

"""
subneuron_df = df[0:5] # choose vertices to use for the subneuron
vertex_list = subneuron_df['sample'].array
print(vertex_list)
"""

[5]:

"\nsubneuron_df = df[0:5] # choose vertices to use for the subneuron\nvertex_list = subneuron_df['sample'].array \nprint(vertex_list)\n"

Download the Volume¶

Download the volume containing the specified vertices.

[6]:

"""
ngl = NeuroglancerSession(url, mip=mip)
buffer = 10
img, bounds, vox_in_img_list = ngl.pull_vertex_list(seg_id, vertex_list, buffer = buffer, expand = True)
"""

[6]:

'\nngl = NeuroglancerSession(url, mip=mip)\nbuffer = 10\nimg, bounds, vox_in_img_list = ngl.pull_vertex_list(seg_id, vertex_list, buffer = buffer, expand = True)\n'

Plot¶

[7]:

"""
# Reference: https://github.com/NeuroDataDesign/mouselit/blob/master/bijan/mouse_test/final%20notebook.ipynb
def napari_viewer(img, labels=None, shapes=None, label_name="Segmentation"):
    viewer = napari.view_image(np.squeeze(np.array(img)))
    if labels is not None:
        viewer.add_labels(labels, name=label_name)
    if shapes is not None:
        viewer.add_shapes(data=shapes, shape_type='path', edge_color='blue', name='Skeleton')
    return viewer
"""

[7]:

'\n# Reference: https://github.com/NeuroDataDesign/mouselit/blob/master/bijan/mouse_test/final%20notebook.ipynb\ndef napari_viewer(img, labels=None, shapes=None, label_name="Segmentation"):\n    viewer = napari.view_image(np.squeeze(np.array(img)))\n    if labels is not None:\n        viewer.add_labels(labels, name=label_name)\n    if shapes is not None:\n        viewer.add_shapes(data=shapes, shape_type=\'path\', edge_color=\'blue\', name=\'Skeleton\')\n    return viewer\n'

Let’s take a look at the downloaded volume. Napari will open in a new window.

[8]:

"""
viewer = napari.Viewer(ndisplay=3)
viewer.add_image(img)
nbscreenshot(viewer)
"""

[8]:

'\nviewer = napari.Viewer(ndisplay=3)\nviewer.add_image(img)\nnbscreenshot(viewer)\n'

[9]:

"""
n=napari_viewer(img)
"""

[9]:

'\nn=napari_viewer(img)\n'

[10]:

"""
import inspect
a = repr(n)
print(a)

b = repr(n).find(('napari.viewer.Viewer'))
print(b)
"""

[10]:

"\nimport inspect\na = repr(n)\nprint(a)\n\nb = repr(n).find(('napari.viewer.Viewer'))\nprint(b)\n"

[11]:

"""
n.window.close()
"""

[11]:

'\nn.window.close()\n'

[12]:

#We get a `corrected_subneuron_df` that contains `(x,y,z)` coordinates within the downloaded volume for the vertices in the SWC.

[13]:

"""
import inspect
a = repr(n)
print(a)

b = repr(n).find(('napari.viewer.Viewer'))
print(b)
"""

[13]:

"\nimport inspect\na = repr(n)\nprint(a)\n\nb = repr(n).find(('napari.viewer.Viewer'))\nprint(b)\n"

[14]:

#We get a `corrected_subneuron_df` that contains `(x,y,z)` coordinates within the downloaded volume for the vertices in the SWC.

[15]:

"""
import inspect
a = repr(n)
print(a)

b = repr(n).find(('napari.viewer.Viewer'))
print(b)
"""

[15]:

"\nimport inspect\na = repr(n)\nprint(a)\n\nb = repr(n).find(('napari.viewer.Viewer'))\nprint(b)\n"

[16]:

# We get a `corrected_subneuron_df` that contains `(x,y,z)` coordinates within the downloaded volume for the vertices in the SWC.

[17]:

"""
import inspect
a = repr(n)
print(a)

b = repr(n).find(('napari.viewer.Viewer'))
print(b)
"""

[17]:

"\nimport inspect\na = repr(n)\nprint(a)\n\nb = repr(n).find(('napari.viewer.Viewer'))\nprint(b)\n"

[18]:

# We get a `corrected_subneuron_df` that contains `(x,y,z)` coordinates within the downloaded volume for the vertices in the SWC.

[19]:

"""
transpose = vox_in_img_list.T
vox_in_img_list_t = transpose.tolist()

corrected_subneuron_df = s3_trace.generate_df_subset(list(vox_in_img_list_t), subneuron_start = 0, subneuron_end = 5)
print(corrected_subneuron_df)
"""

[19]:

'\ntranspose = vox_in_img_list.T\nvox_in_img_list_t = transpose.tolist()\n\ncorrected_subneuron_df = s3_trace.generate_df_subset(list(vox_in_img_list_t), subneuron_start = 0, subneuron_end = 5)\nprint(corrected_subneuron_df)\n'

Convert the SWC to a graph and print some information about the graph.

[20]:

"""
G = s3_trace._df_to_graph(df_voxel=corrected_subneuron_df)
print('Number of nodes:', len(G.nodes))
print('Number of edges:', len(G.edges))
print('Sample 1 coordinates (x,y,z):', G.nodes[1])
paths = s3_trace._graph_to_paths(G)
print('Number of paths:', len(paths))
"""

[20]:

"\nG = s3_trace._df_to_graph(df_voxel=corrected_subneuron_df)\nprint('Number of nodes:', len(G.nodes))\nprint('Number of edges:', len(G.edges))\nprint('Sample 1 coordinates (x,y,z):', G.nodes[1])\npaths = s3_trace._graph_to_paths(G)\nprint('Number of paths:', len(paths))\n"

We can display the SWC on the Volume

[21]:

"""
%gui qt
napari_viewer(img, shapes=paths)
nbscreenshot(viewer)
"""

[21]:

'\n%gui qt\nnapari_viewer(img, shapes=paths)\nnbscreenshot(viewer)\n'

Automatically segment the neuron¶

We start by converting the seed points to a format used by the thresholding.

[22]:

"""
seed = [adaptive_thresh.get_seed(sample)[1] for sample in vox_in_img_list]
print(seed)
"""

[22]:

'\nseed = [adaptive_thresh.get_seed(sample)[1] for sample in vox_in_img_list]\nprint(seed)\n'

Next, we compute a confidence-connected threshold segmentation.

[23]:

"""
labels = adaptive_thresh.confidence_connected_threshold(img, seed, num_iter=1, multiplier=0.5)
"""

[23]:

'\nlabels = adaptive_thresh.confidence_connected_threshold(img, seed, num_iter=1, multiplier=0.5)\n'

We can display the volume, SWC, and segmentation in Napari.

[24]:

"""
%gui qt
viewer = napari_viewer(img, labels=labels, shapes=paths, label_name="Confidence-Connected Threshold")
nbscreenshot(viewer)
"""

[24]:

'\n%gui qt\nviewer = napari_viewer(img, labels=labels, shapes=paths, label_name="Confidence-Connected Threshold")\nnbscreenshot(viewer)\n'

Steps to Manually Edit Labels¶

Labels can be manually edited following these steps:

Ensure Napari is in 2D-slice viewing, not 3D view. (The second button from the bottom left)
Click the image layer and adjust the contrast limits as desired.
Click the “Confidence-Connected Threshold Layer”
Click the paintbrush tool and adjust the brush size. Ensure that “label” is set to 1 to paint and 0 to erase.
Click and drag on the image to adjust labels. Changes are saved automatically, and CMD-Z to undo is supported.

Extract the manual labels for uploading.

[25]:

#manual_labels = viewer.layers['Confidence-Connected Threshold'].data

Upload the segmentation to AWS.

[26]:

#%%capture
#ngl_upload = NeuroglancerSession(url+"_seg", mip=mip)
#ngl_upload.push(manual_labels, bounds);

Confirm that the upload was successful. It was!

[27]:

#downloaded_labels = ngl_upload.pull_bounds_seg(bounds)

[28]:

#print(np.all(manual_labels == downloaded_labels))