Fluorescent immunohistochemistry has typically been limited to a handful of parameters (between 2-6) which does not permit imaging data to sufficiently identify cell subsets. As a result, the technique has been predominantly restricted to the verification of broader findings rather than in true discovery science.
Given the strength of evolutionary pressure exerted upon the immune system by constant conflict with rapidly changing infectious agents across millennia, it is undoubtedly one of the most complex biological systems observed in nature. There are a multitude of highly diverse subsets of immune cells that rapidly alter their behaviour, functionality and composition within a tissue almost continuously.
To better define the nature and function of these immune cell populations high parameter techniques were developed, such as flow cytometry, CyTOF and single-cell RNA-Seq. These have provided much insight but require individual cells to be isolated, destroying the tissue of origin in the process. They therefore miss a fundamental aspect of the underlying biology, namely the precise 3D spatial organisation, local interactions between immune cells and with those of the tissue itself.
Immune responses vary and are heterogenous across tissues including the eye, dependent upon the surrounding vascular, stromal, parenchymal and resident immune cell composition. These differences are obviously critical to understanding pathology as cell localisation and interactions must be distinguished as either central to active destructive lesions or irrelevantly distant.
Our prior work at Dr Ron Germain's group at NIH allowed us to contribute to the development of an open source technique called IBEX, which we can now apply to better study the eye.
We have developed a method to automate the IBEX process and allow a far greater number of samples to be processed in a shorter amount of time. It combines Leica's THUNDER microscope with Fluigent's ARIA microfluidics system and details of this are now published. We are grateful to Moorfields Eye Charity, who are supporting the establishment of this setup in the lab. It will allow us to bring the latest advances in spatial biology to the study of eye disease.
The IBEX Imaging Community is an international group of scientists committed to sharing knowledge related to multiplexed imaging in a transparent and collaborative manner. Links are here to the Knowledge-Base and Zenodo pages. Our group has a commitment to open science, protocol and resource sharing and we are delighted to be part of this community.
This open, global repository is a central resource for reagents, protocols, panels, publications, software, and datasets. In addition to IBEX, we support standard, single cycle multiplexed imaging (Multiplexed 2D imaging), volume imaging of cleared tissues with clearing enhanced 3D (Ce3D), highly multiplexed 3D imaging (Ce3D-IBEX), and extension of the IBEX dye inactivation protocol to the Leica Cell DIVE (Cell DIVE-IBEX).
Mouse retina imaged using IBEX technique
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Updated February 2024