Decoding tissue complexity with hyper-plex microscopy

Tissue and organ function fundamentally depend on the types of cells that constitute them and how they are organised within them. Major advances in single cell transcriptomic analyses have revealed unexpected heterogeneity within cell populations, and it has become all the more important to understand how the spatial relationships between these heterogeneous cell populations influence cell and, ultimately, tissue function. The newly recognised field of spatial biology aims to address this question and was developed thanks to microscopy and tissue preparation advances that allow detecting cells in large 3D tissue preparations. However, microscopy has been traditionally low throughput and up to recently it has only been possible to use immunostainings to identify 4-7 antigens, usually corresponding to 2-3 cell types at a time. Spatial transcriptomics allow genome wide gene expression analyses in situ in 2D; however, they still lack single cell resolution. The gap between traditional microscopy and spatial transcriptomics has now been filled by hyper-plex microscopy, where a number of different protocols have been developed to achieve immunostaining for up to 40 different antigens, followed by combinatorial phenotypic analyses to identify virtually all cell types present in 2D preparations of the tissue of interest, and heterogeneities within such cell populations. Several of these protocols are challenging to implement or require expensive, customised reagents. Here, we have identified a platform composed of a robotic workstation, a high-end microscope and a spatial biology-dedicated software that will take hyper-plex microscopy within reach of a wide and diverse range of researchers across the biomedical field. By working with commercially available, fluorophore conjugated antibodies, users will be able to interrogate tissues for the presence of any combination of markers of interest. Moreover, by uniquely embedding the new technology within two state of the art facilities that support technical apprenticeships, and leveraging on the interdisciplinary culture of Imperial College, we will maximise the impact of this investment and lead in the training of a new generation of quantitative spatial biologists. These researchers will catalyse development of ongoing and future projects to tackle the current challenge of understanding tissue complexity and the cellular and molecular mechanisms underpinning health and disease, leading to the development of improved preventative and therapeutic approaches.

Understanding the link between spatial organisation of heterogeneous cell populations and tissues/organs' function is crucial for the biomedical sciences of the 21st century as it holds the promise to uncover cellular and molecular mechanisms underpinning health and disease, leading to the next generation of improved preventative and therapeutic interventions. Hyper-plex microscopy is the only widely applicable in situ technique capable of providing single cell resolution images reporting on tissue organisation and complexity.

We will implement hyper-plex microscopy through the purchase of a robot-microscope-image analysis software platform that is both highly reliable and extremely flexible. The combination of Leica Cell DIVE Imager with HALO software and BioAssemblyBot 200 Cell DIVE automation workstation was released in summer 2022 and will enable hyper-plex microscopy at Imperial for all users, nucleated by a core of already MRC funded researchers with projects awaiting hyper-plex microscopy capability. The technology will be available to everyone at Imperial and beyond, and its impact will be maximised by the unprecedented opportunity of a two-year technical post and apprenticeship bridged between the well-established FILM facility and the recently formed ICL-Leica Microsystem Imaging Hub. Combined with PI Lo Celso and Co-I Braga's expertise in advanced microscopy and quantitative image analyses and leveraging on the two facilities' mission to bring users and solutions together across disciplines, our vision is that the Leica Cell DIVE platform will enable tackling tissue complexity and catalyse the development of the next generation of quantitative spatial biologists.

The only other equipment capable of hyper-plex immunostainings in the London area are another Leica Cell Dive Imager without robot at King's College, a Codex machine at Crick and a Comet machine at the ICR. All are heavily used, and lack automation, ease of use and throughput, respectively.