Isotropic 3D Digitally Scanned Light Sheet Time-Domain Fluorescence Lifetime Imaging

There has been a recent resurgence in the application of 3-dimensional investigation of complex cell-cell, cell-matrix interactions using tissue organoid cell-culture. This requires novel instrumentation to take advantage of quantitative functional imaging methodologies such as fluorescence resonance energy transfer (FRET) since aberrations and scattering within organoids scale non-linearly with optical pathlength. Methods such as selective plane imaging (SPIM) offer an advantage over confocal methodologies due to their reduced depth of field (increased axial resolution) and large field of view. The Ameer-Beg group recently developed the world's first digitally scanned light sheet fluorescence lifetime imaging (FLIM) platform for imaging functional probes in living Zebrafish but there are significant limitations in the methodology including fluorescence lifetime resolution and sensitivity. Recent advances in detection technology including large arrays of single photon avalanche photodiodes offer potential improvements in single-to-noise and dynamic range as we have shown in both confocal and multiphoton imaging. To date, functional imaging using light sheet methodologies is an unmet technological need. This studentship aims to address this short-fall through development of an instrument which can obtain isotropic resolution over volumes suitable for 3D organotypic cultures. We will apply this novel imaging platform to imaging 3D cell cultures and organoids in direct comparison to well-established 2-photon FLIM. The student will investigate the development of a novel isotropic selective plane imaging technique with fluorescence lifetime imaging capability based on our patent application for Swept array microscopy (UK Patent Application No. 1710743.4). Using a spatial light modulator we will project a complex multibeam array in 3 dimensions into the sample which will be detected in an orthogonal plane using a complementary re-imaging assembly. A cooled SPAD array sensor will be used for detection, acquiring high resolution fluorescence lifetime imaging data with isotropic resolution due to enhanced rejection of scatter with confocal aperturing. As such, this system is unique in having confocal apertures arranged in a Theta microscopy geometry (Stelzer and S. Lindek, Opt. Commun. 111, 536-547 (1994)). Whilst confocal apertures are not required in the 2-photon excitation case, in a theta microscope the single view excitation/detection geometry leads to isotropic resolution through the combination of focused excitation and apertured detection. The concept allows extension to a bidirectional (i.e. reversed excitation/detection) methodology such that isotropic resolution is further improved. The platform offers a unique opportunity to provide interlaced 2-photon en face imaging modes for additional multi-view applications. Biological applications of the new platform can be envisaged in 3D cell culture systems and organoids (Maddy Parsons) to investigate cell substrate interactions. Initially, we will concentrate on imaging of engineered cell matrices with defined mechanical properties using 3D printing technology (EnvisionTEC Bioplotter, Ameer-Beg) to investigate well-understood FRET biosensors such as Rho GTPase and vinculin tension sensors (in which we have considerable expertise), and specialised EPAC sensors (in collaboration with Prof Kees Jalink, Amsterdam) to investigate signalling in response to substrate deformation and molecular cues in 3D.

Milestones:
Year 1
Development of dual digitally scanned light sheet platform with FLIM detection
Software development for instrument control. Transfer of knowledge activities with M Squared.
Year 2
Application of Imaging system to 3D organoids produced using a combination of Bioplotting and microfluidics.
Development of analysis platform for volume rendering of FLIM data in collaboration with M Squared.
Year 3
Investigation