Advances in applied in vivo two-photon fluorescence light-sheet microscopy

Increasingly complicated two-photon light-sheet fluorescence microscopy (LSFM) systems are developed for highly specialised tasks. However, a gap remains in translating such advanced imaging technologies from the research lab to the clinics. Applied technology involves challenging research and engineering tasks, paralleled by rigorous documentation and compliance with standards to gain approval for clinical implementation. Such a process is hampered by any layer of intricacy in the technology.
This dissertation seeks to advance practical solutions in two-photon LSFM to minimise complexity and accelerate clinical translation in the context of disease diagnostics. The research focuses on two primary applications for label-free, two-photon in vivo imaging. (1) Hyperspectral light-sheet microscopy for assessment of functional biomarkers. The goal is a minimally complex hyperspectral light-sheet system that can perform fast, label-free in vivo imaging in live animals. (2) Light-sheet endomicroscopy towards point-of-care colorectal cancer (CRC) diagnostics. This case is part of the European project PROSCOPE. PROSCOPE aims to develop a multimodal imaging probe for in vivo imaging during colonoscopy as a tool for real-time CRC diagnostics. The images must provide real-time in situ information on the morphology of the colon tissue as an optical equivalent to the invasive biopsy.
A two-photon hyperspectral light-sheet microscope was successfully designed, built, and characterised. The results demonstrate the first label-free in vivo imaging in zebrafish using the cosine and sine phasor approach. It was possible to distinguish tissues of different spectra and preliminary identification of metabolic co-factors NADH and FAD, showcasing near-future opportunities for evaluating tissue metabolic activity through the optical redox ratio. The hyperspectral snapshot scheme is relatively simple and shows great potential for implementation into reduced complexity systems for clinical translation. Combined with light-sheet microscopy, it is a powerful tool for fast hyperspectral imaging.
A 1D static Airy LSFM was successfully designed, assembled and characterised. The system offers dynamic control of the Airy light-sheet length and is ready for imaging ex vivo colon tissue to assess sample-specific optimal imaging parameters. The Airy illuminator was designed by the PROSCOPE two-photon team to test the illumination path of the probe. The characterisation revealed a two-photon lightsheet with a field-of-view suitable for colon tissue imaging. The excitation of twophoton fluorescence by the Airy illuminator confirms the illumination path of the fully integrated probe works and shows exciting prospects for the PROSCOPE goal.