Mouse datasets were acquired using the manual IBEX multiplex imaging protocol and accompany the manuscript "IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues", A. Radtke et al., 2020, PNAS.

All image data are stored using the Imaris file format. To view these multi-channel images, you can use one of these free viewers, Imaris viewer, Fiji.

Each experiment has an associated imaging meta-data file in xlsx format and the resulting image in Imaris format.

Mouse spleen (Manual)

Dataset is a 16 parameter IBEX experiment performed on a mouse spleen section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse thymus (Manual)

Dataset is a 26 parameter IBEX experiment performed on a mouse thymus section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse lung (Manual)

Dataset is a 23 parameter IBEX experiment performed on a mouse lung section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.379 µm), y (0.379 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse small intestine (Manual)

Dataset is a 20 parameter IBEX experiment performed on a mouse small intestine section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse liver (Manual)

Dataset is an 18 parameter IBEX experiment performed on a liver section from a LysM-tdtomato reporter mouse labeled with antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse naive lymph node (Manual)

Dataset is a 41 parameter IBEX experiment performed on a mouse lymph node section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Mouse immunized lymph node (Manual)

Dataset is a 41 parameter IBEX experiment performed on a mouse lymph node section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

T cells develop from circulating precursor cells, which enter the thymus and migrate through specialised sub-compartments that support their maturation and selection. In humans, this process starts in early fetal development and is highly active until thymic involution in adolescence. To map the micro-anatomical underpinnings of this process in pre- and early postnatal stages, we established a novel quantitative morphological framework for the thymus, the Cortico-Medullary Axis, and used it to perform a spatially resolved analysis. By applying this framework to a curated multimodal single-cell atlas, spatial transcriptomics, and high-resolution multiplex imaging data, we demonstrate establishment of the lobular cytokine network, canonical thymocyte trajectories and thymic epithelial cell distributions within the first trimester of fetal development. We pinpoint tissue niches of thymic epithelial cell progenitors and distinct subtypes associated with Hassall's corpuscles and uncover divergence in the timing of medullary entry between CD4 vs. CD8 T cell lineages. These findings provide a basis for a detailed understanding of T lymphocyte development and are complemented with a holistic toolkit for cross-platform imaging data analysis, annotation, and Organ Axis construction (TissueTag), which can be applied to any tissue.

40-plex iterative bleaching extends multiplexity (IBEX) imaging was performed on fixed frozen tissues from normal human lymph nodes and follicular lymphoma lymph nodes. Multiplexed immunofluorescence (MxIF) imaging was performed on serial FFPE tissue sections from follicular lymphoma lymph nodes. Four distinct antibody panels were applied to each tissue section referred to as MxIF Panel 1 (MxIF_P1), MxIF Panel 2 (MxIF_P2), MxIF Panel 3 (MxIF_P3), and MxIF Panel 4 (MxIF_P4). Multiplexed imaging was performed on serial FFPE tissue sections from normal and follicular lymphoma lymph nodes using the Cell DIVE imager and IBEX dye inactivation protocol (Cell DIVE-IBEX). Two panels of antibodies, immune (CellDIVE_IBEX_Immune) or stromal (CellDIVE_IBEX_Stromal), were applied to serial sections. IBEX and MxIF imaging were performed on a discovery cohort of 10 samples. Key spatial findings were extended to a larger validation cohort of 29 samples using the Cell DIVE-IBEX method.

These datasets were acquired using either the manual or automated IBEX multiplex imaging protocols and accompany the manuscript "IBEX: An iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues", A. Radtke et al., 2021, Nature Protocols.

All image data are stored using the Imaris file format. To view these multi-channel images, you can either use one of these free viewers, Imaris viewer, Fiji.

Each experiment has an associated imaging meta-data file in xlsx format and the resulting image in Imaris format.

Human Jejunum (Automated)

Dataset is a 24 parameter IBEX experiment performed on a human jejunum section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 µm), y (0.160 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655).

Human Kidney (Automated)

Dataset is a 16 parameter IBEX experiment performed on a human kidney Formalin-Fixed Paraffin-Embedded (FFPE) section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 µm), y (0.160 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655).

Human Lymph Node (Automated)

Dataset is a 25 parameter IBEX experiment performed on a human lymph node section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 µm), y (0.160 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655).

Human Skin (Automated)

Dataset is an 18 parameter IBEX experiment performed on a human skin section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 µm), y (0.160 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655).

Human Liver (Manual)

Dataset is a 22 parameter IBEX experiment performed on a human liver section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Human Lymph Node (Manual)

Dataset is a 38 parameter IBEX experiment performed on a human lymph node section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Human Spleen (Manual)

Dataset is a 25 parameter IBEX experiment performed on a human spleen section labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

These datasets were acquired using the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method described in:

1. "IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues", A. Radtke et al., 2020, doi: 10.1073/pnas.2018488117.
2. "IBEX: An open and extensible method for high content multiplex imaging of diverse tissues", A. Radtke et al., 2021,
   doi: 10.48550/arXiv.2107.11364.

Acquisition of 3D data utilized the manual protocol while acquisition of the 2D data utilized the automated protocol. All data is stored using the Imaris file format.

To view these multi-channel images, you can use one of these free viewers, Imaris viewer (Oxford Instruments), Fiji.

Images can be registered using the code available on GitHub) (XTRegisterSameChannel: "Affine registration of z-stacks using common channel").

A video illustrating the usage of the Imaris extension to register this data is available on YouTube.

Human Spleen

Dataset is comprised of a four-cycle manual IBEX experiment performed on human spleen sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 um), y (0.284 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Markers per channel in each of the cycles:

1. Human_Spleen_Panel1.ims (7 channels): CD21, CD54, Hoechst, CD20, CD68, Glycophorin A, Fibrinogen
2. Human_Spleen_Panel2.ims (7 channels): CD163, CD49a, CD138, Hoechst, CD15, HLA-DR, CD11c
3. Human_Spleen_Panel3.ims (7 channels): Vimentin, CD4, CD31, Ki-67, Hoechst, CD3, CD8
4. Human_Spleen_Panel4.ims (7 channels): SPARC, CD44, Hoechst, CD61, CD45, SMA, Lumican

Registration - If using our Imaris Extensions registration code, this dataset uses the default settings.

Human Liver

Dataset is comprised of a four-cycle manual IBEX experiment performed on human liver sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 um), y (0.284 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Markers per channel in each of the cycles:

1. Human_Liver_Panel1.ims (6 channels): CD54, ASS1, Lyve-1, Hoechst, Keratin 7, CD34
2. Human_Liver_Panel2.ims (6 channels): CD163, CD138, Fibrinogen, Hoechst, HLA-DR, CD68
3. Human_Liver_Panel3.ims (7 channels): CD4, Glutamine Synthetase, Ki-67, Hoechst, CD3, CD8, Tubulin beta 3


4. Human_Liver_Panel4.ims (6 channels): CD44, Vimentin, CD49a, Hoechst, SMA, CD45

Registration - If using our Imaris Extensions registration code, this dataset requires that you modify the default settings. Using the advanced settings dialog you need to:

1. Disable the FFT option.
2. Enable the 2D affine option.

Human Kidney

Dataset is comprised of a four-cycle automated IBEX experiment performed on human kidney Formalin-Fixed Paraffin-Embedded (FFPE) sections labeled with the nuclear marker Hoechst and antibodies directed against the indicated markers. Images were acquired using an inverted Thunder 3D Cell Culture microscope with a high precision (Quantum) stage, a high quantum efficiency sCMOS camera (DFC9000 GTC), and a 40x (1.3) NA oil objective. The light source was an LED8 with 8 individual LED lines for excitation with millisecond triggering. All images were captured at a 16-bit depth with the following pixel dimensions: x (0.160 um), y (0.160 um), and z (1 um). Images were tiled and merged using the LAS X Navigator software (LAS X 3.7.1.21655).

Markers per channel in each of the cycles:

1. Human_Kidney_Panel1.ims (4 channels): Hoechst, Cytokeratin, CD34, SMA
2. Human_Kidney_Panel2.ims (3 channels): Hoechst, CD10, Cathepsin L
3. Human_Kidney_Panel3.ims (3 channels): Hoechst, CD15, TMPRSS2
4. Human_Kidney_Panel4.ims (3 channels): Hoechst, Vimentin, Glycophorin A

Registration - If using our Imaris Extensions registration code, this dataset uses the default settings.

This dataset was acquired using the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method described in: "IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues", A. Radtke et al., 2020, doi:10.1073/pnas.2018488117.

It is comprised of a three cycle IBEX experiment performed on mouse spleen sections labeled with the nuclear marker JOJO-1 and membrane label CD4 AF594. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 mm), y (0.284 mm), and z (1 mm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).

Markers per channel in each of the three cycles:

1. spleen_panel1.nrrd (6 channels): B220 PE, CD8 BV421, IgD AF700, CD4 AF594, JOJO, Foxp3 eF660
2. spleen_panel2.nrrd (7 channels): CD169 PE, F480 BV421, MHCII AF700, CollIV AF488, JOJO, CD11c AF647, CD4 AF594
3. spleen_panel3.nrrd (7 channels): CD31 PE, CD68 BV421, Ki67 AF700, CD45 AF488, CD4 AF594, JOJO, CD3 AF647

The panels can be registered using the code available on github.

To view these multi-channel images, in nrrd format, use the Fiji viewer. The data is stored in XYZC order.

SPACE is an R package for spatial analysis of multiplex images of biological tissues, although spatial data from any context can be used. For many ensembles (i.e. combinations) of spatial elements (e.g. cell types, biomolecules, etc.), SPACE quantifies non-random patterning in a single specimen (called "cisMI") or divergent patterning across groups of specimens (called "transMI"). Thanks to the information-theoretic underpinnings of SPACE, these patterns can be arbitrarily complex: simple co-assortment and mutual exclusion are detected, along with quantitative gradients, structural orientations, context-dependent interactions of 3+ elements, etc. Further tools allow in-depth characterization and visualization of specific ensembles.

SimpleITK based extensions for the imaris (Oxford Instruments) microscopy image analysis program. A variety of utilities including registration, channel arithmetic, virtual H&E staining and more. Extension programs can be run as standalone software without the need for the imaris software.

Software for aligning/registering images acquired with the IBEX microscopy imaging technique. This Python package enables alignment of multiple cycles of fluorescence images, acquired using IBEX. A repeated marker is used to register all panels to a selected panel (in the registration nomenclature this is the fixed image). After registration all panels are resampled onto the fixed image.

While this method was developed for a specific imaging protocol, it will likely work for other protocols that contain a repeated marker. The registration approach is implemented using the SimpleITK toolkit registration framework.

The key implementation aspects include:

1. Multi-phase based approach with robust initialization.
2. Multi-resolution and point sampling.
3. Affine transformation model.
4. Use of correlation as optimized similarity metric.

This program is meant to make advanced data analytic techniques accessible for Histo-Cytometry data. The goal is to take established analytical techniques, such as neural network based clustering algorithms, and package them in a user friendly way, allowing researchers to use them to explore complex datasets.