From 6814aa61b6ec22967850dd7bb2365a24607e3975 Mon Sep 17 00:00:00 2001
From: Ziv Yaniv Mouse datasets were... 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).
These datasets were...
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...
These datasets were acquired using the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method described in:
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:
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:
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:
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:
Registration - If using our Imaris Extensions registration code, this dataset uses the default settings.
These datasets were...
These datasets were acquired using the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method described in:
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:
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:
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:
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:
Registration - If using our Imaris Extensions registration code, this dataset uses the default settings.
This dataset was...
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:
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.