CDeep3M-Plug-and-Play cloud-based deep learning for image segmentation.

Animals were used in accordance with a protocol approved by the Institutional Animal Care and Use Committee at the University of California, San Diego.

Tissue preparation and imaging for serial block-face scanning electron microscopy

(SBEM) and X-ray microscopy (XRM) C57BL/6NHsd mice (Envigo) at age 4-6 weeks were anesthetized using ketamine / xylazine and transcardially perfused with Ringers solution followed by a fixative mix composed of 2.5% glutaraldehyde, 2% formaldehyde, 2 mM CaCl2, in 150mM cacodylate buffer. Fixation was started at 37ºC and cooled to 4ºC during the 15 minute perfusion. The brain was removed, post-fixed in the same fixative mix for 2 hours at 4ºC. Free floating brain sections of 100 μm thickness were prepared in 150mM cacodylate buffer with 2 mM CaCl 2 using a vibratome (Leica), sagittal from the cerebellum and coronal from the lateral habenula for SBEM and coronal from the hippocampus for XRM. The tissue was processed as described in Deerinck et al. 2010 16 . Briefly, sequential staining consisted of 2% OsO 4 / 1.5% potassium ferrocyanide, 0.5% thiocarbohydrazide and OsO 4 , followed by en-bloc uranyl acetate (2%). Sections were dehydrated by a sequence of increasing concentrations of ethanol, followed by dry acetone and then placed into 50:50 Durcupan ACM:acetone overnight. Slices were immersed in 100% Durcupan resin overnight in vacuum, then flat embedded between glass slides and left to harden at 60 °C for 48 hours. SBEM was performed using a Merlin SEM (Zeiss, Oberkochen, Germany) with a Gatan 3View system at high vacuum. The XRM tilt series was collected using a Zeiss Xradia 510 Versa (Zeiss X-Ray Microscopy, Pleasanton, CA, USA) operated at 40 kV (76 μA current) with a 40× magnification and 0.416μm pixel size. XRM volumes were generated from a tilt series of 3201 projections using XMReconstructor (Xradia), resulting in a final reconstructed volume of 391μm × 405μm × 395μm in x/y/z.

Electron tomograms High Pressure Freezing and Freeze Substitution

For high-pressure freezing the brain was removed and post-fixed in the fixative mix for 1 hour at 4ºC. Vibratome sections of 100 μm thickness were transferred into 0.15M cacodylate buffer with 2 mM CaCl 2 before high-pressure freezing. A small portion of the tissue was punched out and placed into a 100 μm deep membrane carrier and surrounded with 20% BSA in 0.15 M cacodylate buffer. The specimens were high pressure frozen with a Leica EM PACT2. Freeze substitution was carried out in extra dry acetone (Acros) as follows: 0.1% tannic acid at −90ºC for 24 hours, wash 3× 20 min in acetone at −90ºC, transferred to 2% osmium tetroxide / 0.1% uranyl acetate and kept at −90ºC for 48 hours, warmed to −60ºC over 15 hours, kept at −60ºC for 10 hours, warmed to 0ºC for 16 hours. The specimens were then washed with ice-cold acetone and allowed to come to room temperature and washed twice more with acetone. The specimens were infiltrated with 1:3 Durcupan ACM resin:acetone for several hours, 1:1Durcupan:acetone for 24 hours, 3:1 Durcupan:acetone for several hours, 100% Durcupan:acetone overnight, and then fresh Durcupan for several hours. The 100% Durcupan steps were done under vacuum. The specimens were then placed in Durcupan in 60ºC oven for 48 hours. The epoxy blocks were cut with a Leica UCT ultramicrotome into 300 nm thick sections. No on-grid staining was performed. Ribbons were collected on slot grids with a 50 nm thick support film (Luxel Corp, Friday Harbor, WA). The grids were glow discharged for 10 seconds on both sides and then coated with 10 nm colloidal gold diluted 1:2 with 0.05M bovine serum albumin solution (Ted Pella, Redding, CA).

Electron tomogram acquisition

Electron tomography was used to digitally reconstruct a small portion (about 1.5μm×1.5μm×1.5μm) of a plastic embedded high-pressure frozen mouse cerebellum specimen at a voxelsize of 1.6nm. The final volume was assembled from 7 consecutive tomograms (serial sections), each generated after a 4-tilt series scheme in which the sample is tilted every 0.5° from −60° to 60° at four distinct azimuthal angles (0°, 90°, 45° and 135°) in an electron beam. The micrographs were acquired on a FEI Titan operating at 300kV with a Gatan Ultrascan 4k×4k CCD camera. An iterative scheme was used during the tomographic processing to reduce the influence of reconstruction artifacts 17 .

Fluorescence microscopy

Tissue was collected at 6 weeks from the cerebellum of C57BL/6NHsd mice (Envigo). Mice were anesthetized using ketamine / xylazine and transcardially perfused with Ringers solution followed by a fixative mix composed of 4% formaldehyde in 1× Phosphate buffered saline (PBS). Fixation was started at 37ºC and cooled to 4ºC during the 30-minute perfusion. The brain was removed, post-fixed in the same fixative mix for 2 hours at 4ºC. Free floating brain sections of 50 μm thickness were prepared using a vibratome (Leica) and stained for 1 hour in 2.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI) in 1× PBS. Sections were mounted using ProLong Gold Antifade Reagent (Molecular Probes) and imaged with the Olympus Fluoview FV1000 confocal laser scanning microscope using 60× magnification lens at a pixel size of 0.21μm in x/y and a step size of 0.3μm in z.

Evaluation of CDeep3M performance

To define the performance of CDeep3M compared to ground truth segmentations, established by human expert annotators, following formulas where used. Pixels or objects are classified in one of four categories, TP: True positive TN: True negative FP: False positive FN: False negative. Precision = TP / (TP+FP); Recall = TP / (TP+FN); F1 value = 2*TP /(2*TP+FP+FN); Jaccard index = TP / (FP + TP + FN) Areas used to evaluate performance did not include areas used for training. Size of training data volumes were as follows in x/y/z: LM: 512×1024×92 voxel; XRM nuclei: 382×974×101 voxel; ET vesicles: 514×514×100 voxel; ET membranes: 1024×1024×20 voxel; ssTEM mitochondria: 1024×768×165 voxel; SBEM nuclei: 1024×1024×15 voxel; SBEM mitochondria: 1024×1024×80 voxel; ssSEM membranes: 1024×1024×100 voxel; SBEM membranes for transfer learning: 1024×1024×20 voxel; SBEM vesicles: 512×512×100 voxel Reporting Summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Data and software availability

CDeep3M source code and documentation are available for download on GitHub ( https://github.com/CRBS/cdeep3m ) and is free for non-profit use. Amazon AWS CloudFormation templates are available with each release enabling easy customization and deployment of CDeep3M for AWS cloud compute infrastructure. For the end user ~10 minutes after creating the CloudFormation stack, a p2x or p3x instance with a fully installed version of CDeep3M will be available to process data. Example data are included in the release. Further data will be made available from the corresponding authors upon reasonable request.