Accompanying dataset for: "IBEX: An iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues"

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).

本数据集采用手动或自动化IBEX多重成像协议采集，配套发表于《Nature Protocols》2021年的论文《IBEX：用于多样组织高内涵成像的迭代免疫标记与化学漂白方法》，作者为A. Radtke等人。

所有图像数据均以Imaris文件格式存储。若要查看这些多通道图像，可使用以下免费查看器之一：Imaris Viewer、Fiji。

每项实验均附带一份xlsx格式的成像元数据文件，以及一份Imaris格式的结果图像文件。

人体空肠（自动化采集）
本数据集为一项基于人体空肠切片的24参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Thunder 3D细胞培养显微镜，搭载高精度（Quantum）载物台、高量子效率sCMOS相机（DFC9000 GTC）以及40×（数值孔径1.3）油浸物镜。光源为LED8，包含8个独立LED通道用于激发，触发精度达毫秒级。所有图像以16位深度采集，像素尺寸如下：x轴0.160 μm，y轴0.160 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.7.1.21655）进行拼接与合并。

人体肾脏（自动化采集）
本数据集为一项基于福尔马林固定石蜡包埋（FFPE）人体肾脏切片的16参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Thunder 3D细胞培养显微镜，搭载高精度（Quantum）载物台、高量子效率sCMOS相机（DFC9000 GTC）以及40×（数值孔径1.3）油浸物镜。光源为LED8，包含8个独立LED通道用于激发，触发精度达毫秒级。所有图像以16位深度采集，像素尺寸如下：x轴0.160 μm，y轴0.160 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.7.1.21655）进行拼接与合并。

人体淋巴结（自动化采集）
本数据集为一项基于人体淋巴结切片的25参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Thunder 3D细胞培养显微镜，搭载高精度（Quantum）载物台、高量子效率sCMOS相机（DFC9000 GTC）以及40×（数值孔径1.3）油浸物镜。光源为LED8，包含8个独立LED通道用于激发，触发精度达毫秒级。所有图像以16位深度采集，像素尺寸如下：x轴0.160 μm，y轴0.160 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.7.1.21655）进行拼接与合并。

人体皮肤（自动化采集）
本数据集为一项基于人体皮肤切片的18参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Thunder 3D细胞培养显微镜，搭载高精度（Quantum）载物台、高量子效率sCMOS相机（DFC9000 GTC）以及40×（数值孔径1.3）油浸物镜。光源为LED8，包含8个独立LED通道用于激发，触发精度达毫秒级。所有图像以16位深度采集，像素尺寸如下：x轴0.160 μm，y轴0.160 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.7.1.21655）进行拼接与合并。

人体肝脏（手动采集）
本数据集为一项基于人体肝脏切片的22参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Leica TCS SP8 X共聚焦显微镜，配备40×物镜（数值孔径1.3）、4个HyD探测器与1个PMT探测器、输出光谱范围为470~670 nm的白光激光器，以及405 nm、685 nm、730 nm分立激光器。所有图像以8位深度采集，行平均次数为3，分辨率为1024×1024，像素尺寸如下：x轴0.284 μm，y轴0.284 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.5.5.19976）进行拼接与合并。

人体淋巴结（手动采集）
本数据集为一项基于人体淋巴结切片的38参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Leica TCS SP8 X共聚焦显微镜，配备40×物镜（数值孔径1.3）、4个HyD探测器与1个PMT探测器、输出光谱范围为470~670 nm的白光激光器，以及405 nm、685 nm、730 nm分立激光器。所有图像以8位深度采集，行平均次数为3，分辨率为1024×1024，像素尺寸如下：x轴0.284 μm，y轴0.284 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.5.5.19976）进行拼接与合并。

人体脾脏（手动采集）
本数据集为一项基于人体脾脏切片的25参数IBEX实验，切片使用核标记物Hoechst以及针对指定靶点的抗体进行标记。图像采集使用倒置Leica TCS SP8 X共聚焦显微镜，配备40×物镜（数值孔径1.3）、4个HyD探测器与1个PMT探测器、输出光谱范围为470~670 nm的白光激光器，以及405 nm、685 nm、730 nm分立激光器。所有图像以8位深度采集，行平均次数为3，分辨率为1024×1024，像素尺寸如下：x轴0.284 μm，y轴0.284 μm，z轴1 μm。图像通过LAS X Navigator软件（LAS X 3.5.5.19976）进行拼接与合并。