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.
Mouse retina imaged using IBEX technique
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Updated December 2022