Reference BioImaging dataset to assess the phenotypic trait diversity of bryophytes within the family Scapaniaceae

Representative voucher specimens were received from herbaria and fresh samples of Diplophyllum taxifolium, Scapania cuspiduligera, Scapania gymnostomophila and Scapania subalpina were additionally collected, put into envelopes on-site, identified and photographed afterwards. Information regarding the date, site (including geographical coordinates), habitat, substrate and other further information are available in the meta-data. For microscopy, a Zeiss Axio Scope.A1 HAL 100/HBO, 6x HD/DIC, M27, 10x/23 microscope with an achromatic-aplanatic 0.9 H D Ph DIC condenser was used with the objectives EC Plan Neofluar 2.5x/0.075 M27 (a=8.8mm), Plan-Apochromat 5x/0.16 M27 (a=12.1mm), Plan-Apochromat 10x/0.45 M27 (a=2.1mm), Plan-Apochromat 20x/0.8 M27 (a=0.55mm), and Plan-Apochromat 40x/0.95 Korr M27 (a=0.25mm) using the EC PN and the Fluar 40x/1.30 III and PA 40x/0.95 III filters for DIC. The conversion filter CB3 and the interference filter wideband green were used to improve digital reproduction of colors. For macroscopy and for preparing microscopy slides, a binocular microscope Zeiss Stemi 2000c was used (apochromatic Greenough system with a stereo angle of 11° and 100/100 switchover of camera and ocular viewing). For stand-alone macroscopic images, the objectives Canon MP-E 65mm 2.8 1-5x macro and Laowa 25mm 2.5-5.0x ultra-macro for Canon EF and the Canon EF-RF adapter. To acquire digital images, a full-frame, high-resolution camera (Canon EOS RP, 26 megapixel) was used and adapted to the microscopes using binocular phototubes with sliding prism 30°/23 (Axio Scope.A1) and 100:0/0:100 reversed image (Stemi 2000c) using 60-T2 camera adapter for Canon EOS and Canon EF-RF adapter. The objectives Canon MP-E and Laowa 25mm were adapted directly through the Canon EF-RF adapter. To construct images with extended depth of field using computational methods, multiple images were recorded at different focal planes. This “focus stacking” approach was automatized for macroscopy by attaching the camera to a Cognisys StackShot macro rail fixed on a Novoflex macro stand, and for microscopy by adapting a Cognisys StackShot motor to the fine adjustment of the microscope using two cogged wheels, one small wheel (1 cm diameter) adapted on the motor and one large wheel (8.5 cm diameter) on the fine adjustment of the microscope. The two cogged wheels were coupled with a toothed belt to obtain very fine step increments of the stepping motor for high magnifications. A Cognisys StackShot controller was used to control the amount and distance of the stepping motor with the following controller settings: Dist/Rev: 3200 stp, Backlash: 0 steps, # pics: 1, Tsettle: 100.0 ms, Toff: 450.0 ms, Auto Return: yes, Speed: 3000 st/sec, Tlapse: off, Tpulse: 800.0 ms, Tramp: 100 ms, Units: steps, Torque: 6, Hi Precision: Off, LCD Backlight: 10, Mode: Auto-Step using between 25 steps (magnification 1x) and 50 steps (magnification 25x) and 100 steps (magnification 400x) (number of steps depending on aperture settings and effective magnification). Raw images were recorded in CR3-format and pre-processed with Adobe Camera RAW. Non-destructive image enhancements such as corrections of the field curvature, removal of chromatic aberration, increase of contrast and brightness were performed in Adobe Camera RAW. Images were then exported into TIF-format and any image enhancement steps were recorded in individual XMP-files. Files containing multiple images of one object captured at different steps of the z-axis (for alter multi-focus image fusion) have been saved into one OME-TIFF file. Multi-focus image fusion was performed on the individual images in the z-stacks using the software Helicon Focus and by choosing the algorithms depth map and pyramid with different settings of radius (4, 8, 16, 24) and smoothing (2, 4). The best composite image was chosen manually and retained. When composite images contained specimen that were larger than the frame, several images were stitched together using the software Affinity Photo and the panorama stitching function. Images were manually segmented and interfering background removed using the flood select, brush selection and freehand selection tools in the software Affinity Photo. In order to determine the scale, a stage micrometre was photographed separately with any of the objectives and microscope combinations. The scale was calculated per pixel for each combination and scale bars were put post-hoc onto the segmented images using a simple Python script. Meta-data including species name, taxonomic rank information, voucher specimen id, image acquisition date, an object description including the name of the captured phenotypic properties, used objective-microscope combination and magnification were associated with any raw image based on unique species names and unique respective file names. Individual file names (variable file list), name within an image focus stack (variable stack name) and name within an image stitching stack (variable stitch name) were recorded additionally to facilitate subsequent automized image processing in workflows. A Python script was created to put individual images as part of image stacks into directories. The Python script parses the Label tag in the XMP-files. Any meta-data regarding image enhancement and non-destructive image processing were extracted from XMP-files using a simple Python script. The meta-data was saved in individual TSV-files and merged using a helper Python script. In order to improve data reuse and to enable linking bioimaging data to ecological data repositories such as GBIF, individual segmented images were associated with standardised geolocation information. Swiss Topo CH1903/LV03 coordinates were converted to WGS84 using Swisstopo-WGS84-LV0352. The segmented images and meta-data contained in a TSV file were uploaded to the Image Data Resource repository (IDR).

Peters K, König-Ries B

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idr0134-peters-bryophytes/experimentA ()

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experimentA ( , parquet: -)

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Data for this study is available at the BioImage Archive: S-BIAD188.

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