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Microscopy

Support for Microscopy was developed as a BIDS Extension Proposal.

Please see Citing BIDS on how to appropriately credit this extension when referring to it in the context of the academic literature.

Microscopy datasets formatted using this specification are available on the BIDS examples repository and can be used for practical guidance when curating a new dataset.

Further Microscopy datasets are available:

Microscopy imaging data

Template:

Legend:
  • For more information about filename elements (for example, entities, suffixes, extensions), follow the links embedded in the filename template.

  • Filename entities or folders between square brackets (for example, [_ses-<label>]) are OPTIONAL.

  • Some entities may only allow specific values, in which case those values are listed in <>, separated by |.

  • _<suffix> means that there are several (>6) valid suffixes for this filename pattern.

  • .<extension> means that there are several (>6) valid extensions for this file type.

  • [.gz] means that both the unzipped and gzipped versions of the extension are valid.

Microscopy data MUST be stored in the micr directory.

File formats

The Microscopy community uses a variety of formats for storing raw data, and there is no single standard that all researchers agree on. However, a standardized file structure has been developed by the Open Microscopy Environment for whole-slide imaging with the OME-TIFF file specifications. The OME-TIFF file allows for multi-page TIFF files to store multiple image planes and supports multi-resolution pyramidal tiled images. An OME-XML data block is also embedded inside the file’s header. Further, OME-ZARR (sometimes referred to as OME-NGFF or NGFF) has been developed to provide improved access and storage for large data via chunked and compressed N-dimensional arrays.

The BIDS standard accepts microscopy data in a number of file formats to accommodate datasets stored in 2D image formats and whole-slide imaging formats, to accommodate lossless and lossy compression, and to avoid unnecessary conversions of the original data from a non-tiled to a tiled format, or vice-versa.

Microscopy raw data MUST be stored in one of the following formats:

If different from PNG, TIFF, OME-TIFF, or OME-ZARR, the original unprocessed data in the native format MAY be stored in the /sourcedata directory.

Modality suffixes

Microscopy data currently support the following imaging modalities:

Name suffix Description
2-photon excitation microscopy 2PE 2-photon excitation microscopy imaging data
Bright-field microscopy BF Bright-field microscopy imaging data
Coherent anti-Stokes Raman spectroscopy CARS Coherent anti-Stokes Raman spectroscopy imaging data
Confocal microscopy CONF Confocal microscopy imaging data
Differential interference contrast microscopy DIC Differential interference contrast microscopy imaging data
Dark-field microscopy DF Dark-field microscopy imaging data
Fluorescence microscopy FLUO Fluorescence microscopy imaging data
Multi-photon excitation microscopy MPE Multi-photon excitation microscopy imaging data
Nonlinear optical microscopy NLO Nonlinear optical microscopy imaging data
Optical coherence tomography OCT Optical coherence tomography imaging data
Phase-contrast microscopy PC Phase-contrast microscopy imaging data
Polarized-light microscopy PLI Polarized-light microscopy imaging data
Scanning electron microscopy SEM Scanning electron microscopy imaging data
Selective plane illumination microscopy SPIM Selective plane illumination microscopy imaging data
Super-resolution microscopy SR Super-resolution microscopy imaging data
Transmission electron microscopy TEM Transmission electron microscopy imaging data
Micro-CT uCT Micro-CT imaging data

Filename entities

In the context of Microscopy, a session (ses-<label>) can refer to all the acquisitions between the start and the end of an imaging experiment for ex vivo imaging, or a subject lab visit for biopsy procedure and/or in vivo imaging. Consistent with other data types in BIDS, the session entity is optional.

The sample-<label> entity is REQUIRED for Microscopy data and is used to distinguish between different samples from the same subject. The label MUST be unique per subject and is RECOMMENDED to be unique throughout the dataset.

For example: Three brain slices (sample-01 to sample-03) extracted from subject sub-01, imaged by scanning electron microscopy (SEM) in PNG format

└─ sub-01/
   └─ micr/
      ├─ sub-01_sample-01_SEM.png 
      ├─ sub-01_sample-02_SEM.png 
      ├─ sub-01_sample-03_SEM.png 
      └─ sub-01_SEM.json 

In this example, the JSON metadata is common for all samples of sub-01. JSON metadata may be defined per subject or per sample as appropriate, as per the inheritance principle.

The acq-<label> entity corresponds to a custom label that MAY be used to distinguish a different set of parameters used for acquiring the same modality. For example, two images of the same sample acquired by bright-field microscopy (BF) in PNG format at different magnification of 40x and 60x. In such case two files could have the following names: sub-01_sample-01_acq-40x_BF.png and sub-01_sample-01_acq-60x_BF.png, however the user is free to choose any other label as long as they are consistent across subjects and sessions.

The stain-<label> entity MAY be used to distinguish image files from the same sample using different stains or antibodies for contrast enhancement.

For example: One brain slice (sample-01) extracted from subject sub-01 with three stains (stain-01, stain-02 and stain-03) in three separate files, imaged by selective plane illumination microscopy (SPIM) in OME-TIFF format

└─ sub-01/
   └─ micr/
      ├─ sub-01_sample-01_stain-01_SPIM.ome.tif 
      ├─ sub-01_sample-01_stain-01_SPIM.json 
      ├─ sub-01_sample-01_stain-02_SPIM.ome.tif 
      ├─ sub-01_sample-01_stain-02_SPIM.json 
      ├─ sub-01_sample-01_stain-03_SPIM.ome.tif 
      └─ sub-01_sample-01_stain-03_SPIM.json 

In this example, the entity stain is used to distinguish images with different stains in separate files from the same sample. In the case where a single file contains different staining in each channel, the stain-<label> is omitted.

Stains SHOULD be indicated in the "SampleStaining" key in the sidecar JSON file, although the label may be different. Description of antibodies SHOULD also be indicated in "SamplePrimaryAntibodies" and/or "SampleSecondaryAntobodies" as appropriate.

If more than one run of the same sample, acquisition and stain are acquired during the same session, the run-<index> entity MUST be used: _run-1, _run-2, _run-3, and so on. If only one run was acquired the run-<index> can be omitted.

The chunk-<index> entity is used when multiples regions (2D images or 3D volumes files) of the same physical sample are imaged with different fields of view, regardless if they overlap or not.

In some cases, the chunks can be "ordered" and, for example, correspond to the displacement of the microscope stage. In other cases, the chunks can be different images of the same sample with no explicit spatial relation between them.

Examples of different chunks configurations can be seen in Figure 1. Figure 1

Figure 1: Examples of chunks configurations.

  • a) ordered 2D chunks without overlap,
  • b) ordered 2D chunks with overlap,
  • c) unordered 2D chunks with and without overlap,
  • d) and e) ordered 2D chunks on different 3D planes,
  • f) ordered 3D chunks.

For example: Four chunks (chunk-01 to chunk-04) from the same brain sample (sample-01) of subject sub-01, imaged by confocal microscopy (CONF) in OME-TIFF format

└─ sub-01/
   └─ micr/
      ├─ sub-01_sample-01_chunk-01_CONF.ome.tif 
      ├─ sub-01_sample-01_chunk-01_CONF.json 
      ├─ sub-01_sample-01_chunk-02_CONF.ome.tif 
      ├─ sub-01_sample-01_chunk-02_CONF.json 
      ├─ sub-01_sample-01_chunk-03_CONF.ome.tif 
      ├─ sub-01_sample-01_chunk-03_CONF.json 
      ├─ sub-01_sample-01_chunk-04_CONF.ome.tif 
      └─ sub-01_sample-01_chunk-04_CONF.json 

The index number can be assigned arbitrarily and, in the case of "ordered" chunks, the chunks' relative positions (in terms of scaling and translation) SHOULD be defined by an affine transformation matrix in the JSON sidecar file of each chunk, as described in Chunk Transformations.

In this example, the JSON metadata is different for each chunk of sub-01_sample-01. JSON metadata may be defined per sample or per chunk as appropriate, as per the inheritance principle.

In microscopy, many pyramidal file formats store multiple resolutions for the same acquisition. In the case where a multiple resolutions file format is converted to single resolution file format, only the higher resolution file is present in the raw data. Lower resolutions files MUST be placed under the derivatives directory and use the res-<label> entity.

For example:

└─ my_dataset/
   ├─ derivatives/
   │  └─ downsampled/
   │     └─ sub-01/
   │        └─ micr/
   │           ├─ sub-01_sample-01_res-4x_TEM.png 
   │           └─ sub-01_sample-01_res-4x_TEM.json 
   └─ sub-01/
      └─ micr/
         ├─ sub-01_sample-01_TEM.png 
         └─ sub-01_sample-01_TEM.json 

See Preprocessed, coregistered and/or resampled volumes for details.

Microscopy metadata (Sidecar JSON)

Microscopy data MUST be described by metadata fields, stored in sidecar JSON files.

Device Hardware

Key name Requirement Level Data type Description
Manufacturer RECOMMENDED string Manufacturer of the equipment that produced the measurements.
ManufacturersModelName RECOMMENDED string Manufacturer's model name of the equipment that produced the measurements.
DeviceSerialNumber RECOMMENDED string The serial number of the equipment that produced the measurements. A pseudonym can also be used to prevent the equipment from being identifiable, so long as each pseudonym is unique within the dataset.
StationName RECOMMENDED string Institution defined name of the machine that produced the measurements.
SoftwareVersions RECOMMENDED string Manufacturer's designation of software version of the equipment that produced the measurements.
InstitutionName RECOMMENDED string The name of the institution in charge of the equipment that produced the measurements.
InstitutionAddress RECOMMENDED string The address of the institution in charge of the equipment that produced the measurements.
InstitutionalDepartmentName RECOMMENDED string The department in the institution in charge of the equipment that produced the measurements.

Image Acquisition

Key name Requirement Level Data type Description
PixelSize REQUIRED array of numbers A 2- or 3-number array of the physical size of a pixel, either [PixelSizeX, PixelSizeY] or [PixelSizeX, PixelSizeY, PixelSizeZ], where X is the width, Y the height and Z the depth. If the file format is OME-TIFF, these values need to be consistent with PhysicalSizeX, PhysicalSizeY and PhysicalSizeZ OME metadata fields, after converting in PixelSizeUnits according to PhysicalSizeXunit, PhysicalSizeYunit and PhysicalSizeZunit OME fields.
PixelSizeUnits REQUIRED string Unit format of the specified "PixelSize". MUST be one of: "mm" (millimeter), "um" (micrometer) or "nm" (nanometer).

Must be one of: "mm", "um", "nm".
Immersion OPTIONAL string Lens immersion medium. If the file format is OME-TIFF, the value MUST be consistent with the Immersion OME metadata field.
NumericalAperture OPTIONAL number Lens numerical aperture (for example: 1.4). If the file format is OME-TIFF, the value MUST be consistent with the LensNA OME metadata field.

Must be a number greater than 0.
Magnification OPTIONAL number Lens magnification (for example: 40). If the file format is OME-TIFF, the value MUST be consistent with the "NominalMagnification" OME metadata field.

Must be a number greater than 0.
ImageAcquisitionProtocol OPTIONAL string Description of the image acquisition protocol or URI (for example from protocols.io).
OtherAcquisitionParameters OPTIONAL string Description of other relevant image acquisition parameters.

Sample

Key name Requirement Level Data type Description
BodyPart RECOMMENDED string Body part of the organ / body region scanned. From DICOM Body Part Examined (for example "BRAIN").
BodyPartDetails RECOMMENDED string Additional details about body part or location (for example: "corpus callosum").
BodyPartDetailsOntology OPTIONAL string URI of ontology used for BodyPartDetails (for example: "https://www.ebi.ac.uk/ols/ontologies/uberon").
SampleEnvironment RECOMMENDED string Environment in which the sample was imaged. MUST be one of: "in vivo", "ex vivo" or "in vitro".

Must be one of: "in vivo", "ex vivo", "in vitro".
SampleEmbedding OPTIONAL string Description of the tissue sample embedding (for example: "Epoxy resin").
SampleFixation OPTIONAL string Description of the tissue sample fixation (for example: "4% paraformaldehyde, 2% glutaraldehyde").
SampleStaining RECOMMENDED string or array of strings Description(s) of the tissue sample staining (for example: "Osmium"). MAY be an array of strings if different stains are used in each channel of the file (for example: ["LFB", "PLP"]).
SamplePrimaryAntibody RECOMMENDED string or array of strings Description(s) of the primary antibody used for immunostaining. Either an RRID or the name, supplier and catalogue number of a commercial antibody. For non-commercial antibodies either an RRID or the host-animal and immunogen used (for examples: "RRID:AB_2122563" or "Rabbit anti-Human HTR5A Polyclonal Antibody, Invitrogen, Catalog # PA1-2453"). MAY be an array of strings if different antibodies are used in each channel of the file.
SampleSecondaryAntibody RECOMMENDED string or array of strings Description(s) of the secondary antibody used for immunostaining. Either an RRID or the name, supplier and catalogue number of a commercial antibody. For non-commercial antibodies either an RRID or the host-animal and immunogen used (for examples: "RRID:AB_228322" or "Goat anti-Mouse IgM Secondary Antibody, Invitrogen, Catalog # 31172"). MAY be an array of strings if different antibodies are used in each channel of the file.
SliceThickness OPTIONAL number Slice thickness of the tissue sample in the unit micrometers ("um") (for example: 5).

Must be a number greater than 0.
TissueDeformationScaling OPTIONAL number Estimated deformation of the tissue, given as a percentage of the original tissue size (for examples: for a shrinkage of 3%, the value is 97; and for an expansion of 100%, the value is 200).

Must be a number greater than 0.
SampleExtractionProtocol OPTIONAL string Description of the sample extraction protocol or URI (for example from protocols.io).
SampleExtractionInstitution OPTIONAL string The name of the institution in charge of the extraction of the sample, if different from the institution in charge of the equipment that produced the image.

Chunk Transformations

Chunk transformations metadata describes the spatial relation between chunks of the same sample in an implicit coordinate system.

  • The source frame of reference is the frame of reference of the associated image.

  • The target frame of reference is the implicit coordinate system of the transform.

  • The target frame of reference has the same units as the PixelSizeUnits metadata.

  • The chunk transformation is described by 2 metadata fields: an affine transformation matrix and a description of the axis of the matrix.

  • Other transformations should be described in derivatives.

Key name Requirement Level Data type Description
ChunkTransformationMatrix RECOMMENDED if chunk-<index> is used in filenames array of arrays 3x3 or 4x4 affine transformation matrix describing spatial chunk transformation, for 2D and 3D respectively (for examples: [[2, 0, 0], [0, 3, 0], [0, 0, 1]] in 2D for 2x and 3x scaling along the first and second axis respectively; or [[1, 0, 0, 0], [0, 2, 0, 0], [0, 0, 3, 0], [0, 0, 0, 1]] in 3D for 2x and 3x scaling along the second and third axis respectively). Note that non-spatial dimensions like time and channel are not included in the transformation matrix.
ChunkTransformationMatrixAxis REQUIRED if ChunkTransformationMatrix is present array of strings Describe the axis of the ChunkTransformationMatrix (for examples: ["X", "Y"] or ["Z", "Y", "X"]).

An example of chunk transformations JSON metadata for chunk-01 and chunk-05 of Figure 2 is shown below:

Figure 2

Figure 2: Example figure for chunks transformations.

In this example, there is no scaling and chunk-01 is at the origin. chunk-05 is translated by 5 um along X+ and by 3 um along Y+.

*_chunk-01_<suffix>.json:

{
        "PixelSize": [1, 1],
        "PixelSizeUnits": "um",
        "ChunkTransformationMatrix": [[1, 0, 0],
                                      [0, 1, 0],
                                      [0, 0, 1]],
        "ChunkTransformationMatrixAxis": ["X", "Y"]
}

*_chunk-05_<suffix>.json:

{
        "PixelSize": [1, 1],
        "PixelSizeUnits": "um",
        "ChunkTransformationMatrix": [[1, 0, 5],
                                      [0, 1, 3],
                                      [0, 0, 1]],
        "ChunkTransformationMatrixAxis": ["X", "Y"]
}

Example of sidecar JSON file (*_<suffix>.json)

{
        "Manufacturer": "Hamamatsu",
        "ManufacturersModelName": "C9600-12",
        "PixelSize": [0.23, 0.23],
        "PixelSizeUnits": "um",
        "Magnification": 40,
        "BodyPart": "BRAIN",
        "BodyPartDetails": "corpus callosum",
        "SampleEnvironment": "ex vivo",
        "SampleFixation": "4% paraformaldehyde, 2% glutaraldehyde",
        "SampleStaining": "LFB",
        "SliceThickness": 5,
        "TissueDeformationScaling": 97
}

Required Samples file

For Microscopy data, the Samples file samples.tsv is REQUIRED and its associated sidecar samples.json file is RECOMMENDED.

Additional optional columns MAY be used to describe other samples' attributes.

For Microscopy data, we RECOMMEND to make use of the columns species, strain and strain_rrid in the Participants file when applicable.

Additional optional columns MAY be used to describe other subjects' attributes.

participants.tsv example:

participant_id species strain strain_rrid
sub-01 mus musculus C57BL/6J RRID:IMSR_JAX:000664
sub-02 mus musculus C57BL/6J RRID:IMSR_JAX:000664

participants.json example:

{
    "species": {
        "Description": "binomial species name from the NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi)"
    },
    "strain": {
        "Description": "name of the strain of the species"
    },
    "strain_rrid": {
        "Description": "research resource identifier (RRID) of the strain (https://scicrunch.org/resources/Organisms/search)"
    }
}

Photos of the samples (*_photo.<extension>)

Photos of the tissue sample, overview microscopy scans or blockface images from cutting MAY be included for visualization of large samples or to indicate the location of chunks in a sample.

Template:

Legend:
  • For more information about filename elements (for example, entities, suffixes, extensions), follow the links embedded in the filename template.

  • Filename entities or folders between square brackets (for example, [_ses-<label>]) are OPTIONAL.

  • Some entities may only allow specific values, in which case those values are listed in <>, separated by |.

  • _<suffix> means that there are several (>6) valid suffixes for this filename pattern.

  • .<extension> means that there are several (>6) valid extensions for this file type.

  • [.gz] means that both the unzipped and gzipped versions of the extension are valid.

The file <extension> for photos MUST be either .jpg, .png or .tif.

The acq-<label> entity MAY be used to indicate acquisition of different photos of the same sample.

For example:

└─ sub-01/
   └─ ses-01/
      └─ micr/
         ├─ sub-01_ses-01_sample-01_acq-1_photo.jpg 
         └─ sub-01_ses_01_sample-01_acq-2_photo.jpg 

Photo data MAY be accompanied by a JSON file containing the following fields. The IntendedFor field is used to link the photo to specific image(s) it was acquired for.

Key name Requirement Level Data type Description
PhotoDescription OPTIONAL string Description of the photo.
IntendedFor OPTIONAL string or array The paths to files for which the associated file is intended to be used. Contains one or more BIDS URIs. Using forward-slash separated paths relative to the participant subdirectory is DEPRECATED. This field is OPTIONAL, in case the photos do not correspond to any particular images, it does not have to be filled.

For example: sub-01_ses-01_sample-01_acq-1_photo.json

{
   "PhotoDescription": "After clearing",
   "IntendedFor": [
        "ses-01/micr/sub-01_ses-01_sample-01_run-1_chunk-01_SPIM.ome.tif",
        "ses-01/micr/sub-01_ses-01_sample-01_run-1_chunk-02_SPIM.ome.tif",
        "ses-01/micr/sub-01_ses-01_sample-01_run-1_chunk-03_SPIM.ome.tif",
        "ses-01/micr/sub-01_ses-01_sample-01_run-1_chunk-04_SPIM.ome.tif"
    ]
}

Below is an example of a spinal cord SEM overview, modified from Zaimi et al., 2018. doi:10.1038/s41598-018-22181-4.

    sub-01_sample-01_photo.jpg
SEM overview