Globus3D light-sheet microscopy data for SELMA3D 2024 challenge - Training subset with no annotations - whole brain images
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- 1 Ludwig-Maximilians-Universität München
- 2 Imperial College London
- 3 Helmholtz Zentrum München
AccessionS-BIAD1197
DescriptionThis dataset is the training set containing whole-brain images without annotations for the SELMA3D challenge. The SELMA3D challenge focuses on self-supervised learning for 3D light-sheet microscopy image segmentation. Its objective is to encourage the development of self-supervised learning methods for general segmentation of various structures in 3D light-sheet microscopy images. The dataset contains 3D whole-brain microscopy image of different labeled biological structures, including blood vessels, c-Fos labeled brain cells involved in neural activity, cell nuclei, and Alzheimer's disease plaques.
Keywordslight-sheet microscopy,
fluorescence microscopy,
3D microscopic image,
image segmentation,
deep learning
LicenseCC BY 4.0
Publication
Self-supervised learning for 3D light-sheet microscopy image segmentation
doi
10.5281/zenodo.10991463
Biosamples for whole-brain blood vessel images
OrganismMus musculus (mouse)
DescriptionMice were housed under a 12-h light/12-h dark cycle for 3 months.
Biological entitymouse brain
Biosamples for brain cell nucleus images
OrganismHomo sapiens (human)
DescriptionHuman brain was taken from human body donor with no known neuropathological diseases.
Biological entityhuman brain
Biosamples for whole-brain c-Fos-positive cell images
OrganismMus musculus (mouse)
DescriptionMale BALB/c mice were housed under a 12-h light/12-h dark cycle and fed a regular unrestricted chow diet for 10–12 weeks.
Biological entitymouse brain
Biosamples for whole-brain amyloid plaque images
OrganismMus musculus (mouse)
Description5xFAD mouse mice were housed under a 12-h light/12-h dark cycle.
Biological entitymouse brain
Specimens for whole-brain blood vessel images
Sample preparation protocol150μl (2%(vol/vol) in saline) EB dye (Sigma-Aldrich, E2129) was injected intraperitoneally into 3-mothold male mice from the C57BL/6J, CD1 and BALB/c strains (Charles River, strain codes 027, 482 and 028, respectively). Twelve hours afer injection of EB dye, we anesthetized the animals with a combination of midazolam, medetomidine and fentanyl (administered intraperitoneally; 1ml per 100 g body weight containing 5mg, 0.5mg and 0.05mg per kg body weight, respectively) and opened their chest for transcardial perfusion. Medium with WGA (0.25mg WGA conjugated to Alexa Fluor 594 dye (Termo Fisher Scientifc, W11262) in 150µl PBS, pH7.2) was supplied by peristaltic pump set to deliver the medium at a rate of 8ml/min, along with 15ml of 1× PBS and 15ml of 4% paraformaldehyde. This short perfusion protocol was established on the basis of preliminary experiments, where both WGA and EB staining were partially washed out, with the goal of delivering fixative to brain tissue via the vessels to achieve a homogenous preservation efect.
After perfusion, brains were extracted from the neurocranium while severing some of the segments of the circle of Willis, which is an inevitable component of most retrieval processes aside from corrosion cast techniques. Next, the samples were incubated in 3DISCO clearing solutions. Briefly, we immersed them in a gradient of tetrahydrofuran (Sigma-Aldrich, 186562): 50%, 70%, 80% and 90% (in distilled water) followed by 100%, at 25 °C for 12h at each concentration. At this point, we modified the protocol by incubating the samples in tert-butanol for 12h at 35 °C followed by immersion in dichloromethane (Sigma-Aldrich, 270997) for 12h at room temperature and a final incubation with refractive index-matched BABB solution (benzyl alcohol+benzyl benzoate, 1:2; Sigma-Aldrich, 24122 and W213802), for at least 24h at room temperature until transparency was achieved. Each incubation step was carried out on a laboratory shaker.
Please refer to https://www.nature.com/articles/s41592-020-0792-1 for more details about the preparation step.
After perfusion, brains were extracted from the neurocranium while severing some of the segments of the circle of Willis, which is an inevitable component of most retrieval processes aside from corrosion cast techniques. Next, the samples were incubated in 3DISCO clearing solutions. Briefly, we immersed them in a gradient of tetrahydrofuran (Sigma-Aldrich, 186562): 50%, 70%, 80% and 90% (in distilled water) followed by 100%, at 25 °C for 12h at each concentration. At this point, we modified the protocol by incubating the samples in tert-butanol for 12h at 35 °C followed by immersion in dichloromethane (Sigma-Aldrich, 270997) for 12h at room temperature and a final incubation with refractive index-matched BABB solution (benzyl alcohol+benzyl benzoate, 1:2; Sigma-Aldrich, 24122 and W213802), for at least 24h at room temperature until transparency was achieved. Each incubation step was carried out on a laboratory shaker.
Please refer to https://www.nature.com/articles/s41592-020-0792-1 for more details about the preparation step.
Specimens for brain cell nucleus images
Sample preparation protocolThe brains of human males were fixed in situ by whole head perfusion via carotid and vertebral arteries under a pressure of below 1 bar. The head was first perfused with 5 L heparinized 0.01 M PBS (10 U/ml of heparin, Ratiopharm), followed by 3 L 4% PFA in 0.01 M PBS for 2-3 h. The veins were finally closed to maintain the PFA to the brain. Then the brains were recovered by calvarian dissection and preserved at least 1-2 weeks for post-fixation submersed in the 4% PFA solution.
PFA-fixed intact human brain was actively pumped with 5 L 10% w/v CHAPS and 25% w/v N- Methyldiethanolamine solution for one month with solution refreshed once at day 15. Then the solution was changed to PBS for active washing for 2 days. The intact human brain was cooled in PBS at 4 °C overnight, then directly cut into 1.5 cm-thick slices in coronal plane using a Rotation Cutting Slicer (Rotation Schneidemaschine, Biodur, Heidelberg, Germany). The total 12 slices were serially labeled and stored in 70% EtOH at 4 °C.
A 1.5 cm-thick intact human brain slice was randomly chosen and passively incubated with 400 mL TO-PRO-3 (1:2000 dilution) in PBS at room temperature for 1 week. Then the solution was changed to 400 mL with 100 mM of Methoxy X-04 (Tocris, 4920) in 40% EtOH (pH = 10 adjusted by NaOH (Roth, 6771.1)) and the sample was incubated for another week. After labeling, the slice was washed with PBS for 1 day. The clearing started with dehydration using a series of 1 L of EtOH/DiH2O solutions (50%, 70%, 100%, 100% v/v) and followed by delipidation using 1 L DCM. Each step lasted 1 day. Then the samples were incubated in 1 L BABB solution at room temperature until completely transparency in around 2 weeks.
1.5 cm-thick human brain slices were randomly chosen and dehydrated with a series of 1 L EtOH/DiH2O (50%, 70%, 100%, 100% v/v), then delipidated with 2 L DCM/MeOH (2:1 v/v), then rehydrated with a series of 1 L EtOH/DiH2O (100%, 70%, 50%, 0% v/v) at room temperature. After incubating with 1 L 0.5 M acetic acid (Roth, T179.1) in DiH2O, this solution was changed to a mixture of 4 M guanidine hydrochloride (Roth, 6069.3), 0.05 M sodium acetate (Sigma, S2889) and 2% Triton X-100 in PBS, pH = 6.0, at room temperature to loosen the extra cellular matrix. The incubation time for each of all above mentioned solutions was 1 day. Next, the slices were shortly incubated with 10% w/v CHAPS and 25% w/v N-Methyldiethanolamine solution for 4 hours and washed with PBS for 1 day. The intact slices were stored in the blocking buffer (0.2% Triton X-100/10% DMSO (Roth, A994.2)/10% goat serum/PBS) at 4 °C.
Regions of interest (including hippocampus, primary motor cortex, primary sensory cortex, and primary visual cortex, 2-4 cm x 2-4 cm x 1.5 cm) were cut and incubated with the same blocking buffer at 37 °C for 1 day. Then the samples were incubated with rabbit antibody anti-Iba1 (1:1000, Wako, 019-19741) or rabbit antibody anti-TH (1:250, abcam, ab112) in the antibody incubation buffer (3% goat serum/3% DMSO/0.2% Tween-20/10mg∙L-1Heparin/PBS) for 1 week at 37 °C. After the primary antibody incubation, samples were washed with the washing buffer (0.2% Tween-20/10mg∙L-1Heparin/PBS) for 1 day refreshing 3 times and then incubated with Alexa 647-conjugated secondary antibodies (1:500, Thermo Fisher, A-21245) in the antibody incubation buffer for 1 week at 37 °C. Other samples were incubated with DyLight 649-lectin (1:500, Vector, DL-1178) in the antibody incubation buffer for 1 week at 37 °C. After washing with PBS, propidium iodide (1:100, Thermo Fisher, P3566) or TO-PRO-3 dye was added in PBS for 3 days at 37 for cell nuclei staining. After labeling, the samples were dehydrated with a series of solutions of EtOH/DiH2O (50%, 70%, 100%, 100% v/v) and delipidated with the DCM solution for 4 hours each solution followed by BABB incubation at room temperature until the sample transparency was reached in 2-3 days.
Please refer to https://www.sciencedirect.com/science/article/pii/S0092867420301112 for more details about the preparation step.
PFA-fixed intact human brain was actively pumped with 5 L 10% w/v CHAPS and 25% w/v N- Methyldiethanolamine solution for one month with solution refreshed once at day 15. Then the solution was changed to PBS for active washing for 2 days. The intact human brain was cooled in PBS at 4 °C overnight, then directly cut into 1.5 cm-thick slices in coronal plane using a Rotation Cutting Slicer (Rotation Schneidemaschine, Biodur, Heidelberg, Germany). The total 12 slices were serially labeled and stored in 70% EtOH at 4 °C.
A 1.5 cm-thick intact human brain slice was randomly chosen and passively incubated with 400 mL TO-PRO-3 (1:2000 dilution) in PBS at room temperature for 1 week. Then the solution was changed to 400 mL with 100 mM of Methoxy X-04 (Tocris, 4920) in 40% EtOH (pH = 10 adjusted by NaOH (Roth, 6771.1)) and the sample was incubated for another week. After labeling, the slice was washed with PBS for 1 day. The clearing started with dehydration using a series of 1 L of EtOH/DiH2O solutions (50%, 70%, 100%, 100% v/v) and followed by delipidation using 1 L DCM. Each step lasted 1 day. Then the samples were incubated in 1 L BABB solution at room temperature until completely transparency in around 2 weeks.
1.5 cm-thick human brain slices were randomly chosen and dehydrated with a series of 1 L EtOH/DiH2O (50%, 70%, 100%, 100% v/v), then delipidated with 2 L DCM/MeOH (2:1 v/v), then rehydrated with a series of 1 L EtOH/DiH2O (100%, 70%, 50%, 0% v/v) at room temperature. After incubating with 1 L 0.5 M acetic acid (Roth, T179.1) in DiH2O, this solution was changed to a mixture of 4 M guanidine hydrochloride (Roth, 6069.3), 0.05 M sodium acetate (Sigma, S2889) and 2% Triton X-100 in PBS, pH = 6.0, at room temperature to loosen the extra cellular matrix. The incubation time for each of all above mentioned solutions was 1 day. Next, the slices were shortly incubated with 10% w/v CHAPS and 25% w/v N-Methyldiethanolamine solution for 4 hours and washed with PBS for 1 day. The intact slices were stored in the blocking buffer (0.2% Triton X-100/10% DMSO (Roth, A994.2)/10% goat serum/PBS) at 4 °C.
Regions of interest (including hippocampus, primary motor cortex, primary sensory cortex, and primary visual cortex, 2-4 cm x 2-4 cm x 1.5 cm) were cut and incubated with the same blocking buffer at 37 °C for 1 day. Then the samples were incubated with rabbit antibody anti-Iba1 (1:1000, Wako, 019-19741) or rabbit antibody anti-TH (1:250, abcam, ab112) in the antibody incubation buffer (3% goat serum/3% DMSO/0.2% Tween-20/10mg∙L-1Heparin/PBS) for 1 week at 37 °C. After the primary antibody incubation, samples were washed with the washing buffer (0.2% Tween-20/10mg∙L-1Heparin/PBS) for 1 day refreshing 3 times and then incubated with Alexa 647-conjugated secondary antibodies (1:500, Thermo Fisher, A-21245) in the antibody incubation buffer for 1 week at 37 °C. Other samples were incubated with DyLight 649-lectin (1:500, Vector, DL-1178) in the antibody incubation buffer for 1 week at 37 °C. After washing with PBS, propidium iodide (1:100, Thermo Fisher, P3566) or TO-PRO-3 dye was added in PBS for 3 days at 37 for cell nuclei staining. After labeling, the samples were dehydrated with a series of solutions of EtOH/DiH2O (50%, 70%, 100%, 100% v/v) and delipidated with the DCM solution for 4 hours each solution followed by BABB incubation at room temperature until the sample transparency was reached in 2-3 days.
Please refer to https://www.sciencedirect.com/science/article/pii/S0092867420301112 for more details about the preparation step.
Specimens for whole-brain c-Fos-positive cell images
Sample preparation protocolMice were killed following deep anesthesia with a mix of ketamine/xylazine, followed by intracardiac perfusion with heparinized PBS (10 U/ml heparin) and by a perfusion with 4% paraformaldehyde (PFA). Tissues and organs were dissected, weighed and post-fixed at 4 °C overnight. Immunostaining for c-Fos was performed using a modified version of SHANEL (https://www.sciencedirect.com/science/article/pii/S0092867420301112). All incubation steps were carried out under moderate shaking (300 rpm). For the pretreatment, samples were dehydrated with an ethanol/water series (50%, 70% and 100% ethanol) at room temperature for 3 h per step. Next, samples were incubated in dichloromethane (DCM) /methanol (2:1 v/v) at room temperature for 1 day. Brains were rehydrated with an ethanol/water series (100%, 70% and 50% ethanol and diH2O) at room temperature for 3 h per step. Samples were incubated in 0.5 M acetic acid at room temperature for 5 h followed by washing with diH2O. Next, brains were incubated in 4 M guanidine HCl, 0.05 M sodium acetate, 2% v/v Triton X-100, pH 6.0, at room temperature for 5 h followed by washing with diH2O. Brains were incubated in a mix of 10% CHAPS and 25% N-methyldiethanolamine at 37 °C for 12 h before washing with diH2O. Blocking was performed by incubating the brains in 0.2% Triton X-100, 10% dimethylsulfoxide and 10% goat serum in PBS shaking at 37 °C for 2 days. Samples were incubated with c-Fos primary antibody (Cell Signaling Technology, 2250, 1:1000 dilution) in primary antibody buffer (0.2% Tween-20, 5% dimethylsulfoxide, 3% goat serum and 100 µl heparin per 100 ml PBS) shaking at 37 °C for 7 days. The antibody solution was filtered (22-µm pore size) before use. Samples were washed in washing solution (0.2% Tween-20 and 100 µl heparin in 100 ml PBS) shaking at 37 °C for 1 day at which the washing solution was refreshed five times. Brains were incubated with the secondary antibody (Alexa Fluor 647 and goat anti-rabbit IgG (H + L) from Invitrogen, A-21245, 1:500 dilution) in secondary antibody buffer (0.2% Tween-20, 3% goat serum and 100 µl heparin per 100 ml PBS) shaking at 37 °C for 7 days followed by incubating in washing solution shaking at 37 °C for 1 day at which the washing solution was refreshed five times. Brains were dehydrated using 3DISCO with a THF/H2O series (50%, 70%, 90% and 100% THF) for 12 h per step followed by an incubation in DCM for 1 h. Tissues were incubated in benzyl alcohol/benzyl benzoate (1:2 v/v) until tissue transparency was reached (>4 h).
Please refer to https://www.nature.com/articles/s41592-024-02245-2 for more details about the preparation step.
Please refer to https://www.nature.com/articles/s41592-024-02245-2 for more details about the preparation step.
Specimens for whole-brain amyloid plaque images
Sample preparation protocolMice were anesthetized using a combination of midazolam, medetomidine and fentanyl (MMF) (1mL/100g of body mass for mice; i.p.). As soon as the animals did not show any pedal reflex, they were intracardially perfused with cold heparinized 0.1 M PBS (10 U/mL of Heparin, Ratiopharm; 100–125 mmHg pressure using a Leica Perfusion One system) for 5–10 min at room temperature until the blood was washed out, followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) (Sigma) for 10 min. Then, the brains were extracted and post-fixed in 4% PFA for 1 day at 4 °C and later washed with 0.1 M PBS for 10 min 3 times at room temperature. For the collection of fresh frozen samples, animals were sacrificed by cervical dislocation and brains were quickly snapped frozen in liquid nitrogen and stored in −80 °C until further processing. Whole brains were dehydrated with gradual addition of methanol in PBS (50% x1, 80% x1, 100% x2, each for 1 h). Overnight bleaching with 5% hydrogen peroxide in methanol was done at 4 °C. Brains were then gradually rehydrated in 100%, 80%, 50% methanol in PBS (1 h for each step, followed by 2 additional washes in PBS). Detergent washing was then performed in PBS with 0.2% Triton X-100 for 2 h, brains were incubated overnight at 37 °C in PBS with 0.2% Triton X-100 and 0.3 M glycine, followed by blocking in PBS with 0.2% Triton X-100 and 6% goat serum for 7 days. Following blocking, the tissue was washed for 1 h twice in PBS with 0.2% Tween 20 and 10 μg/mL heparin (PTwH). Next, brains were incubated with 10 μM Congo Red (Sigma, C6277) at 37 °C in PTwH for 5 days. After that, brains were washed in PTwH for 2 days with periodic solution changes and gradually dehydrated using 3DISCO clearing as described next. Dissected brains were placed in 5 mL tubes (Eppendorf, 0,030,119.401) and covered with 4.5 mL of clearing solution. All incubation steps were performed in a fume hood with gentle shaking or rotation, with the samples covered with aluminum foil to keep them in dark. To clear the samples using 3DISCO, gradient of tetrahydrofuran (THF) in distilled water (v/v %), 2 h for each step, was used as 50%, 70%, 90%, 100% and overnight 100% THF; after dehydration, the samples were incubated for 45 min in dichloromethane (DCM, Sigma, 270,997), and finally in BABB (benzyl alcohol + benzyl benzoate 1:2, Sigma, 24,122 and W213802) until transparency.
Please refer to https://www.cell.com/cell/fulltext/S0092-8674(22)01465-9 for more details about the preparation step.
Please refer to https://www.cell.com/cell/fulltext/S0092-8674(22)01465-9 for more details about the preparation step.
Image acquisition - blood vessel images
Imaging instrumentImaging was performed using a 4× objective lens (Olympus XLFLUOR 340) equipped with an immersion corrected dipping cap mounted on a LaVision UltraII microscope coupled to a white-light laser module (NKT SuperK Extreme EXW-12) for imaging.
Image acquisition parametersImages were taken with 16-bit depth and at a nominal resolution of 1.625 μm per voxel on the x and y axes. In the z dimension, we took sectional images in 3-μm steps while using left- and right-sided illumination.
Imaging methodfluorescence microscopy
Image acquisition - cell nucleus images
Imaging instrumentImaging was performed using a 12x objective lens ( LaVision BioTec MI PLAN) coupled to an Olympus revolving zoom body unit (U-TVCAC) kept at 1x.
Image acquisition parametersImages were taken with 16-bit depth and at a nominal resolution of 0.54 μm per voxel on the x and y axes. In the z dimension, we took sectional images in 5-μm steps.
Imaging methodfluorescence microscopy
Image acquisition - c-Fos-positive cell images
Imaging instrumentImaging was performed using a 4× objective lens (Olympus XLFLUOR 340) equipped with an immersion corrected dipping cap mounted on a LaVision UltraII microscope coupled to a white-light laser module (NKT SuperK Extreme EXW-12) for imaging.
Image acquisition parametersImages were taken with 16-bit depth and at a nominal resolution of 1.625 μm per voxel on the x and y axes. In the z dimension, we took sectional images in 6-μm steps while using left- and right-sided illumination.
Imaging methodfluorescence microscopy
Image acquisition - amyloid plaque images
Imaging instrumentImaging was performed using a 4× objective lens (Olympus XLFLUOR) coupled to an Olympus revolving zoom body unit (U-TVCAC) kept at 1x.
Image acquisition parametersImages were taken with 16-bit depth and at a nominal resolution of 1.63 μm per voxel on the x and y axes. In the z dimension, we took sectional images in 4-μm steps.
Imaging methodfluorescence microscopy
Study Component
Namewhole-brain blood vessel images
Description9 whole-brain blood vessel images. Every image has two channels, C00 for WGA (mainly microvessels) and C01 for EB (mainly major blood vessels).
File Listvessel_samples.json
Associations
Study Component
Namebrain cell nucleus images
Description4 brain cell nucleus images respectively of the hippocampus, motor cortex, sensory cortex, visual cortex.
File Listcell_nucleus_samples.json
Associations
Study Component
Namewhole-brain c-Fos-positive cell images
Description18 whole-brain c-Fos-positive cell images.
File Listc-Fos_brain_cell_samples.json
Associations
Study Component
Namewhole-brain amyloid plaque images
Description4 whole-brain amyloid plaque images.
File ListAb_plaque_samples.json
Associations