Our team has pioneered the development of intravital imaging technology – a microscope with the ability to look directly into living tissues in real-time – which offers the exciting prospect of studying the complex cancer–immune cell ecosystem in toto for the first time.
We aim to build the next generation of intravital two-photon microscopes explicitly designed to image cancer-immune cell interactions in their native microenvironment – the NICHEscopes. We have designed different and complementary types of NICHEscopes to achieve two specific objectives. The core NICHEscope technologies are a revolutionary Raster Adaptive Optics (RAO) module and a Leica deep in vivo explorer microscope with fast fluorescence lifetime imaging technology. Together, these two bespoke next-generation NICHEscopes will enable multimodal, multiscale imaging of deep tissues that has not been possible until now.
The Molecular NICHEscope is a Leica SP8 DIVE (deep in vivo explorer) system with a novel fluorescence lifetime acquisition module, the FALCON (FAstLifetime CONtrast). It is a customised system specifically suited to longitudinal in vivo imaging that requires deep tissue penetration and precise fluorescence unmixing. The molecular NICHEscope is the only spectrally tuneable multiphoton microscope on the market with specialised optics to significantly increase the penetration depth of the imaging volume. This is particularly import for co-localisation analysis of cell to cell and subcellular protein interactions. In combination with the high resolution tandem galvanometric and high-speed resonant scanner, very high scan speeds reduce phototoxicity and makes it suitable for imaging rapidly occurring cellular events as well as for fast large area imaging. This is very relevant for in vivo imaging of lung and abdominal windows to directly observe cellular responses to combination therapies.
The Leica FALCON unit combines TCSPC (time-correlated single photon counting) with fast data handling and analysis algorithms to achieve high acquisition speeds. FLIM data can be acquired for 3D stacks, large mosaic-tiling and time-lapse imaging without compromising on signal intensity, imaging speed and resolution. This results in a higher image stability which would normally be impacted by native sample movement such as respiration and heartbeat. The combination of novel optics and data analysis tools makes it the perfect system for novel deep tissue imaging on a sub cellular level using titanium windows to allow direct imaging of lungs, pancreas and liver, organotypic 3D assays and long-term cellular studies.
The multi-modal imaging approach will allow the integration cell migration and signalling events to the outcomes of cellular interactions, for example between CTLs, NK cells and cancer cells. The Molecular NICHEscope will also enable intravital imaging of drug bioavailability and action and in situ target validation for its preclinical drug testing pipeline. In addition, the implementation of the custom-written Galene image stabilisation software (Palmer Innovation Prize) will allow us to image tumours in moving tissue such as the lung in a breathing animal. The Galene-enabled Molecular NICHEscope will allow us to monitor bottleneck events such as extravasation into secondary sites such as the liver and lung due to in vivo motion artefacts from the beating heart and ventilating lung in live animals and the impact of stromal targeting on the recruitment of CTLs, their motility and ability to seek and destroy cancer targets.
The EndoNICHEscope encompassing a custom-built RAO module will allow us to image larger volumes at greater speed, resolution and depth than possible with conventional two-photon microscopes. In turn, this represents a quantum gain in imaging capability and will enable us, for the first time, to dynamically see cells and molecules in the 'dark space' below the surface of tumours and deep inside tissues in ways that have not been seen before. This is critical because immune cells such as CD8+ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are highly motile cells that migrate in and out of tumours. Immune surveillance involves bidirectional signalling between receptors expressed by these immune cells and ligands expressed by cancer cells (see research question 2, below). In addition, the RAO module will allow us to shape the laser beam and focus it through opaque tissue such as intact bone, enabling us to perform intravital two-photon photoconversion to tag unknown cells in the cancer niche optically. We can then identify them and their transcriptional state by scRNA-seq.
