As part of wider effort towards FAIR data, submissions to the BioImage Archive should feature REMBI-compliant metadata. Below are the main pieces of information required when making a REMBI submission.

If your data requires more information in order to be Findable, Accessible, Interoperable and Reusable, contact us to discuss your use-case.

REMBIStudy

study*
Description
Type Study
Required true
study_components*
Description
Type List[ StudyComponent ]
Required true
sample*
Description
Type List[ Biosample ]
Required true
specimen*
Description
Type List[ Specimen ]
Required true
image_acquisition*
Description
Type List[ ImageAcquisition ]
Required true
image_correlation
Description
Type ImageCorrelation
Required false
image_analysis
Description
Type ImageAnalysis
Required false

Study

General study information
title*
Description The title for your dataset. This will be displayed when search results including your data are shown. Often this will be the same as an associated publication.
Type FreeText
Required true
Validation Unicode; Minimum length: 25 characters
Examples

Visualization of loop extrusion by DNA nanoscale tracing in single cells

SARS-COV-2 drug repurposing - Caco2 cell line

Large-scale electron microscopy database for human type 1 diabetes

description*
Description Use this field to describe your dataset. This can be the abstract to an accompanying publication.
Type FreeText
Required true
Validation Unicode; Minimum length: 25 characters
keywords*
Description Keywords describing your data that can be used to aid search and classification.
Type FreeText
Required true
Examples

RNA localisation

CRISPR

Brain

authors*
Description
Type List[ Author ]
Required true
license
Description
Type License
Required false
funding
Description
Type Funding
Required false
publications
Description
Type List[ Publication ]
Required false
links
Description
Type List[ Link ]
Required false
acknowledgements
Description Any people or groups that should be acknowledged as part of the dataset.
Type FreeText
Required false

Author

last_name*
Description Author last name.
Type FreeText
Required true
first_name*
Description Author first name.
Type FreeText
Required true
email
Description Author e-mail address.
Type FreeText
Required false
orcid
Description Author ORCID ID.
Type FreeText
Required false
affiliation*
Description
Type OrganisationInfo OR OrganisationURL
Required true
role
Description Author role in the study.
Type FreeText
Required false

OrganisationInfo

name*
Description The name of the organisation.
Type FreeText
Required true
address
Description The address of the organisation.
Type FreeText
Required false

OrganisationURL

URL to a public registry containing organisation information. ROR recommended
url*
Description URL
Type FreeText
Required true

License

The license under which the data are available.

Funding

funding_statement*
Description A description of how the data generation was funded.
Type FreeText
Required true
grant_references
Description
Type List[ GrantReference ]
Required false

GrantReference

identifier*
Description The identifier for the grant.
Type FreeText
Required true
funder*
Description The funding body provididing support.
Type FreeText
Required true

Publication

title*
Description Title of associated publication.
Type FreeText
Required true
authors
Description Authors of associated publication.
Type FreeText
Required false
doi
Description Digital Object Identifier (DOI).
Type FreeText
Required false
year
Description Year of publication.
Type FreeText
Required false
pubmed_id
Description PubMed identifier for the publication.
Type FreeText
Required false

StudyComponent

name*
Description The name of your study component.
Type FreeText
Required true
Examples

Experiment A

Screen B

Stitched max-projected fluorescent confocal images

description*
Description An explanation of your study component.
Type FreeText
Required true

Biosample

organism*
Description
Type Organism
Required true
biological_entity*
Description What is being imaged.
Type FreeText
Required true
Examples

Adult mouse corpus callosum

Drosophila endoderm

AC16s human cardiomyoctye cells

description
Description High level description of sample.
Type FreeText
Required false
Examples

Bronchial epithelial cell culture

intrinsic_variables
Description Intrinsic (e.g. genetic) alteration.
Type FreeText
Required false
Examples

stable overexpression of HIST1H2BJ-mCherry and LMNA

Jurkat E6.1 transfected with emerald-VAMP7

Homozygous GFP integration into mitotic genes

extrinsic_variables
Description External treatment (e.g. reagent).
Type FreeText
Required false
Examples

Plate-bound anti-CD3 activation

2-(9-oxoacridin-10-yl)acetic acid

cridanimod

experimental_variables
Description What is intentionally varied between multiple images.
Type FreeText
Required false
Examples

Time

Genotype

Light exposure

Organism

Species.
scientific_name*
Description Scientific name.
Type FreeText
Required true
Examples

Homo sapiens

Arabidopsis thaliana

Danio rerio

common_name
Description Common name.
Type FreeText
Required false
Examples

human

thale cress

zebrafish

ncbi_taxon*
Description NCBI Taxon for the organism.
Type FreeText
Required true
Examples

http://purl.obolibrary.org/obo/NCBITaxon_9606

http://purl.obolibrary.org/obo/NCBITaxon_3702

http://purl.obolibrary.org/obo/NCBITaxon_7955

Specimen

How the sample was grown or cultured and prepared for imaging.
sample_preparation*
Description How the sample was prepared for imaging.
Type FreeText
Required true
Examples

Cells were cultured on poly-L-lysine treated coverslips. Culture media was aspirated, and coverslips were washed once with PBS. Cells were fixed by incubating for 10 min with 4 % formaldehyde/PBS, washed twice with PBS, and permeabilized by incubating (>3 h, -20°C) in 70 % ethanol. Cells were rehydrated by incubating (5 min, RT) with FISH wash buffer (10 % formamide, 2x SSC). For hybridization, coverslips were placed cell-coated side down on a 48μl drop containing 100 nM Quasar570-labelled probes complementary to one of REV-ERBα, CRY2, or TP53 transcripts (Biosearch Technologies) (see Table S6 for probe sequences), 0.1 g/ml dextran sulfate, 1 mg/ml E. coli tRNA, 2 mM VRC, 20 μg/ml BSA, 2x SSC, 10 % formamide and incubated (37°C, 20 h) in a sealed parafilm chamber. Coverslips were twice incubated (37°C, 30 min) in pre-warmed FISH wash buffer, then in PBS containing 0.5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) (5 min, RT), washed twice with PBS, dipped in water, air-dried, placed cell-coated side down on a drop of ProLong Diamond Antifade Mountant (Life Technologies), allowed to polymerize for 24 h in the dark and then sealed with nail varnish.

Immunostained spreads of Arabidopsis pachytene cells were prepared for 3D-SIM imaging as follows. To roughly stage the meiocytes, a single anther from a floral bud was removed and squashed in a drop of water on a clean slide under a coverslip and inspected using brightfield microscopy. Early- and mid-pachytene meiocytes were still stuck together within a meiocyte column, whilst late-pachytene meiocytes had begun to break apart from one-another. More precise staging of early and late pachytene meiocytes was also based on previously defined HEI10 behaviour, with mid pachytene meiocytes exhibiting an intermediate phenotype. The remaining 5 anthers containing meiocytes of the desired stage were dissected from the staged buds. They were then macerated using a brass rod on a No. 1.5H coverslip (Marienfeld) in 10 µl digestion medium (0.4% cytohelicase, 1.5% sucrose, 1% polyvinylpyrolidone in sterile water) for 1 min. Coverslips were then incubated in a moist chamber at 37 °C for 4 min before adding 10 µl of 2% lipsol solution followed by 20 µl 4% paraformaldehyde (pH 8). Coverslips were dried in the fume hood for 3 h, blocked in 0.3% bovine serum albumin in 1x phosphate-buffered saline (PBS) solution and then incubated with primary antibody at 4 °C overnight and secondary antibody at 37 °C for 2 h. In between antibody incubations, coverslips were washed 3 times for 5 min in 1x PBS plus 0.1% Triton X-100. Coverslips were then incubated in 10 µl DAPI (10 µg/ml) for 5 min, washed and mounted on a slide in 7 µl Vectashield.

Cells grown on coverslips were fixed in ice-cold methanol at _20 _ C for 10 min. After blocking in 0.2% gelatine from cold-water fish (Sigma) in PBS (PBS/FSG) for 15 min, coverslips were incubated with primary antibodies in blocking solution for 1h. Following washes with 0.2% PBS/FSG, the cells were incubated with a 1:500 dilution of secondary antibodies for 1 h (donkey anti- mouse/rabbit/goat/sheep conjugated to Alexa 488 or Alexa 594; Molecular Probes or donkey anti-mouse conjugated to DyLight 405, Jackson ImmunoResearch). The cells were counterstained with 1 _g ml_1 Hoechst 33342 (Sigma) to visualize chromatin. After washing with 0.2% PBS/FSG, the coverslips were mounted on glass slides by inverting them into mounting solution (ProLong Gold antifade, Molecular Probes). The samples were allowed to cure for 24-48 h.

growth_protocol
Description How the specimen was grown, e.g. cell line cultures, crosses or plant growth.
Type FreeText
Required false
Examples

Cells were cultured on poly-L-lysine treated coverslips. Culture media was aspirated, and coverslips were washed once with PBS. Cells were fixed by incubating for 10 min with 4 % formaldehyde/PBS, washed twice with PBS, and permeabilized by incubating (>3 h, -20°C) in 70 % ethanol. Cells were rehydrated by incubating (5 min, RT) with FISH wash buffer (10 % formamide, 2x SSC). For hybridization, coverslips were placed cell-coated side down on a 48μl drop containing 100 nM Quasar570-labelled probes complementary to one of REV-ERBα, CRY2, or TP53 transcripts (Biosearch Technologies) (see Table S6 for probe sequences), 0.1 g/ml dextran sulfate, 1 mg/ml E. coli tRNA, 2 mM VRC, 20 μg/ml BSA, 2x SSC, 10 % formamide and incubated (37°C, 20 h) in a sealed parafilm chamber. Coverslips were twice incubated (37°C, 30 min) in pre-warmed FISH wash buffer, then in PBS containing 0.5 μg/ml 4’,6-diamidino-2-phenylindole (DAPI) (5 min, RT), washed twice with PBS, dipped in water, air-dried, placed cell-coated side down on a drop of ProLong Diamond Antifade Mountant (Life Technologies), allowed to polymerize for 24 h in the dark and then sealed with nail varnish.

Immunostained spreads of Arabidopsis pachytene cells were prepared for 3D-SIM imaging as follows. To roughly stage the meiocytes, a single anther from a floral bud was removed and squashed in a drop of water on a clean slide under a coverslip and inspected using brightfield microscopy. Early- and mid-pachytene meiocytes were still stuck together within a meiocyte column, whilst late-pachytene meiocytes had begun to break apart from one-another. More precise staging of early and late pachytene meiocytes was also based on previously defined HEI10 behaviour, with mid pachytene meiocytes exhibiting an intermediate phenotype. The remaining 5 anthers containing meiocytes of the desired stage were dissected from the staged buds. They were then macerated using a brass rod on a No. 1.5H coverslip (Marienfeld) in 10 µl digestion medium (0.4% cytohelicase, 1.5% sucrose, 1% polyvinylpyrolidone in sterile water) for 1 min. Coverslips were then incubated in a moist chamber at 37 °C for 4 min before adding 10 µl of 2% lipsol solution followed by 20 µl 4% paraformaldehyde (pH 8). Coverslips were dried in the fume hood for 3 h, blocked in 0.3% bovine serum albumin in 1x phosphate-buffered saline (PBS) solution and then incubated with primary antibody at 4 °C overnight and secondary antibody at 37 °C for 2 h. In between antibody incubations, coverslips were washed 3 times for 5 min in 1x PBS plus 0.1% Triton X-100. Coverslips were then incubated in 10 µl DAPI (10 µg/ml) for 5 min, washed and mounted on a slide in 7 µl Vectashield.

Cells grown on coverslips were fixed in ice-cold methanol at _20 _ C for 10 min. After blocking in 0.2% gelatine from cold-water fish (Sigma) in PBS (PBS/FSG) for 15 min, coverslips were incubated with primary antibodies in blocking solution for 1h. Following washes with 0.2% PBS/FSG, the cells were incubated with a 1:500 dilution of secondary antibodies for 1 h (donkey anti- mouse/rabbit/goat/sheep conjugated to Alexa 488 or Alexa 594; Molecular Probes or donkey anti-mouse conjugated to DyLight 405, Jackson ImmunoResearch). The cells were counterstained with 1 _g ml_1 Hoechst 33342 (Sigma) to visualize chromatin. After washing with 0.2% PBS/FSG, the coverslips were mounted on glass slides by inverting them into mounting solution (ProLong Gold antifade, Molecular Probes). The samples were allowed to cure for 24-48 h.

ImageAcquisition

imaging_method*
Description
Type ImagingMethod
Required true
imaging_instrument*
Description Description of the instrument used to capture the images.
Type FreeText
Required true
Examples

Zeiss Elyra PS1

Luxendo MuVi SPIM light-sheet microscope

DeltaVision OMX V3 Blaze system (GE Healthcare) equipped with a 60x/1.42 NA PlanApo oil immersion objective (Olympus), pco.edge 5.5 sCMOS cameras (PCO) and 405, 488, 593 and 640 nm lasers

image_acquisition_parameters*
Description How the images were acquired, including instrument settings/parameters.
Type FreeText
Required true
Examples

Two and three days after surgery, sparse labeling of OPCs was achieved by i.p. injection of Tamoxifen (Tam; 180mg/kg bodyweight). Imaging fields of view containing identified OPCs were selected to obtain 10-20 SPOTs per mouse. Chronic 2-photon imaging was performed starting 3 days after surgery. All SPOTs were checked on a daily basis and no z-stack was acquired if no changes occurred. The imaging was performed on custom-built 2-photon microscope (Sutter Instrument Movable Objective Microscope type) using a long-working distance objective (water immersion, 16x magnification, 0.8NA, Nikon) and equipped with a ytterbium-doped laser system at 1045nm and 200fs (Femtotrain, High-Q lasers) or a fiber oscillator 45 laser at 1070nm (Fidelity-2, Coherent) to excite tdTomato labeled cells in the CC. Emission light was detected using a photomultiplier tube (Hamamatsu) after passing a red emission filter (610/75 nm; AHF).

Embryos were imaged on a Luxendo MuVi SPIM light-sheet microscope, using 30x magnification setting on the Nikon 10x/0.3 water objective. The 488 nm laser was used to image nuclei (His-GFP), and the 561 nm laser was used to image transcriptional dots (MCP-mCherry), both at 5% laser power. Exposure time for the green channel was 55 ms and exposure for the red channel was 70 ms. The line illumination tool was used to improve background levels and was set to 40 pixels.

Spherical aberration was minimized using immersion oil with refractive index (RI) 1.514. 3D image stacks were acquired over the whole nuclear volume in z and with 15 raw images per plane (five phases, three angles). The raw data were computationally reconstructed with SoftWoRx 6.5.2 (GE Healthcare) using channel-specific OTFs recorded using immersion oil with RI 1.512, and Wiener filter settings between 0.002-0.006 to generate 3D stacks of 115 nm (488 nm) or 130 nm (593 nm) lateral and approximately 350 nm axial resolution. Multi-channel acquisitions were aligned in 3D using Chromagnon software (Matsuda et al, 2018) based on 3D-SIM acquisitions of multi-colour EdU-labelled C127 cells(Kraus et al, 2017).

ImagingMethod

What method was used to capture images.
value*
Description The text description of the ontology entry.
Type FreeText
Required true
Examples

bright-field microscopy

spinning disk confocal microscopy

high-voltage electron microscopy (HVEM)

ontology_name*
Description The name of the ontology.
Type FreeText
Required true
Examples

Biological Imaging Methods Ontology (FBbi)

ontology_id*
Description The URI identifier for the ontology value.
Type FreeText
Required true
Examples

http://purl.obolibrary.org/obo/FBbi_00000243

http://purl.obolibrary.org/obo/FBbi_00000253

http://purl.obolibrary.org/obo/FBbi_00000622

ImageCorrelation

How images from the same correlative study are linked
spatial_and_temporal_alignment*
Description Method used to correlate images from different modalities
Type FreeText
Required true
Examples

Manual overlay

Alignment algorithm

fiducials_used*
Description Features from correlated datasets used for colocalisation
Type FreeText
Required true
transformation_matrix*
Description Correlation transforms
Type FreeText
Required true

ImageAnalysis

analysis_overview*
Description How image analysis was carried out.
Type FreeText
Required true
Examples

Image segmentation was performed for each 2D slice using a program called ilastik, which utilizes semantic segmentation. 3D object creation from 2D binary images and feature extraction was performed in a program called Arivis.

Each 3D-SIM image contained one nucleus (in a small number of cases multiple nuclei were present, which did not affect the analysis). The image analysis pipeline contained six main steps: bivalent skeleton tracing, trace fluorescence intensity quantification, HEI10 peak detection, HEI10 foci identification, HEI10 foci intensity quantification, and total bivalent intensity quantification. Note that the normalization steps used for foci identification differ from those used for foci intensity quantification; the former was intended to robustly identify foci from noisy traces, whilst the latter was used to carefully quantify foci HEI10 levels.

Images were deconvolved using the default conservative deconvolution method using DeltaVision Softworx software. Image quantification was carried out using Fiji (Schindelin et al, 2012). Deconvolved images were compressed to 2D images displaying the maximum intensity projection for each pixel across z-stacks listed in Table S7 (column “Projected”). Cell and nuclear areas were outlined using thresholding functions on the background TRITC signal and DAPI signal, respectively. Dots corresponding to transcripts were then counted for both nuclear and cytoplasmic areas for each image by applying the “Find Maxima” command with a noise tolerance specified in Table S7 (column “Maxima”). Bar charts show the mean number of dots per nuclear area and cytoplasmic area across all images for all combined replicates.