REL 2025-06-16 2025-09-05 2025-09-05 0009-0002-1572-598X Kohki Konishi Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London Great Maze Pond London United Kingdom SE1 1UL Kohki Konishi Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London Great Maze Pond London United Kingdom SE1 1UL 0009-0002-1572-598X Kohki Konishi Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London Great Maze Pond London United Kingdom SE1 1UL 0000-0003-1542-6218 Roland Fleck Centre for Ultrastructural Imaging, King’s College London, London, UK Great Maze Pond London United Kingdom SE1 1UL Konishi KK Neves GN Russell MR Burrone JB Fleck RF Wellcome Trust 215508/Z/19/Z United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) BB/Z514962/1 United Kingdom Royal Society INF\R2\202061 United Kingdom 2.5 10.6019/EMPIAR-12850 CLEM tissue Konishi KK Neves GN Russell MR Mimura MM Burrone JB Fleck RF Journal of Microscopy J. Microsc United Kingdom 10.1111/jmi.13436 S-BIAD2077 Transmission electron micrograph and segmentation /data/EM2B6 micrographs - single frame TIFF TIFF 1 1 UNSIGNED 16 BIT INTEGER variable variable variable variable

TEM Imaging and Data Acquisition Section TEM Mapping Entire sections were imaged using a Jeol JEM-1400 Flash TEM at an acceleration voltage of 80 kV. Limitless Panorama montaging software was used to acquire typical montages of 400×400 µm2 with a pixel size of 340 nm (X/Y). Region of Interest (ROI) TEM Mapping High-resolution images of regions around the ROI were acquired using the same TEM parameters as for section mapping, except the typical montage size was 70×100 µm2 with a pixel size of 3.4 nm (X/Y). TEM Tomography For 3D reconstruction, regions around the ROI were imaged using a Jeol JEM-1400 Flash TEM at an acceleration voltage of 120 kV. Tilt series were acquired using SerialEM.9 The typical reconstructed volume was 4×4×0.2 µm3 with a voxel size of 1.8 nm. Tomograms were reconstructed using IMOD software. File Naming Convention Files are named according to the following structure: {ExperimentID}_{AcquisitionDate}_{Microscope}_{SampleType}_{PixelSize}.tif - ExperimentID: Unique identifier for the experiment. - AcquisitionDate: Date of image acquisition (YYMMDD). - Microscope: Name of the microscope used for acquisition. -- JEM1400: Jeol JEM-1400 Flash TEM - SampleType: Describes the grid and section. -- Slot{GridBoxPosition}{Grid}{Section}: e.g., Slot812 indicates Grid Box Position 8, Grid 1, Section 2. - PixelSize: Resolution of the image. -- xy{Value}nm: For 2D images, e.g., xy340nm for a pixel size of 340 nm. Examples: Section TEM map: 266_221116_JEM1400_Slot812_xy340nm.tif ROI TEM map: 266_240222_JEM1400_Slot812_xy3.4nm.tif TEM Tomography: 266_240515_JEM1400_Slot813_xy1.8nm.tif TEM Tomography: 266_240530_JEM1400_Slot733_xy1.8nm.tif Filename and description EM2B6/266_221116_JEM1400_Slot812_xy170nm.tif "Section TEM map for Fig.5A" EM2B6/266_240326GN_JEM1400_Slot812_xy3.4nm_img.zarr "ROI TEM map for Figs.5D and 5E" EM2B6/266_240515_JEM1400_Slot813_xy1.8nm.tif "TEM tomogram for Fig.5" EM2B6/266_240515_JEM1400_Slot813_xy3.4nm_z45_img.tif "TEM tomogram for Fig.5F-ii" EM2B6/266_240530_JEM1400_Slot733_xy3.4nm_x33_y0_z43-60.tif "TEM tomogram for Fig.5F-i" EM2B6/266_240530_JEM1400_Slot733_xy3.4nm_x33_y0_z53_img.tif "TEM tomogram for Fig.5F-i" EM2B6/266_240530_JEM1400_Slot733_xy3.4nm_x33_y0_z53_seg.tif "TEM tomogram for Fig.5F-i" EM2B6/266_240530_JEM1400_Slot733_xy1.8nm.tif "TEM tomogram for Fig.5" EM2B6/266_240621GN_JEM1400_Slot721_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot722_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot723_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot731_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot732_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot733_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot734_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot812_xy136nm_x187_y294_img.tif "Section TEM map - cell and nuclei segmentation for Fig.5D" EM2B6/266_240621GN_JEM1400_Slot812_xy136nm_x187_y294_labels.tif "Section TEM map - cell and nuclei segmentation for Fig.5D" EM2B6/266_240621GN_JEM1400_Slot812_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot813_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot814_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot821_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot822_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot823_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B" EM2B6/266_240621GN_JEM1400_Slot824_xy136nm_x187_y294_seg.tif "Section TEM map - nuclei segmentation for Fig.5B"

data/EM2B6 The filenames adhere to a structured naming convention that includes: Experiment ID (266): An experiment-specific ID. Acquisition Date (e.g., 221116, 240326): Indicates when the image was captured or processed (YYMMDD format). Microscope (JEM1400): Specifies the use of a Jeol JEM-1400 Flash TEM. Sample Type (Slot###): Denotes the specific grid and section number, e.g., "Slot812" for Grid Box Position 8, Grid 1, Section 2. Pixel/Voxel Size (xy###nm): Represents the resolution of the image in nanometers. Additional Modifiers (e.g., _img, _seg, _labels, _z##, _x##_y##): These provide further details about the file content: _img: Raw image data. _seg: Segmented data, often showing specific structures like nuclei. _labels: Labeled image data, usually associated with segmentation. _z##, _x##, _y##: Indicate specific slice numbers or coordinates within a 3D volume, particularly for tomograms. .tif or .zarr: File format, where .tif is a common image format and .zarr is a cloud-native, chunked array format suitable for large datasets.