Quantitative Multidimensional Imaging: A Centre of Excellence for Fluorescence Lifetime Imaging Microscopy

Confocal and Multiphoton microscopy have become the mainstay of biological imaging since their commercial introduction in the 80's and 90's respectively. As they have become more established the level of sophistication available to biological researchers has grown to the extent that even experimental techniques considered at the extreme forefront of microscopy (such as super-resolution microscopy and in vivo imaging) have become relatively straightforward. Despite this, functional imaging methods such as fluorescence lifetime imaging microscopy remain challenging outside specialist labs. Fluorescence lifetime imaging (FLIM) is a key microscopy technique used to measure local variations in biological cells and tissues. It is used as an image contrast mechanism to report on many characteristics within the cellular environment and are essential to understanding the complexities of biological systems - King's is currently world leading in this area. Unfortunately, the barriers for FLIM adoption are high due to the complexity of both the technology and analysis procedures and, development time to implement assays. To remain at the forefront of research at the interface between technology development and bioimaging we recognise that these facilities must be equitably available to all researchers in the field. To achieve this, we propose to purchase a Leica Stellaris 8 Dive Falcon to create a centre of excellence for fluorescence lifetime imaging to be located within the Microscope Innovation Centre at King's. The Falcon represents the current commercial state-of-the art for FLIM, multiphoton and confocal imaging, is highly appropriate to support the proposed science and will be highly complementary to cutting-edge developments occurring within the MIC including hyper-spectral FLIM, light-sheet, super-resolution and high content screening (Poland, Ameer-Beg). By co-locating the Falcon with these advanced imaging platforms in an open access, centrally managed core facility we aim to democratise the use of FLIM across a far broader base of biological samples and encourage the use of this powerful technique in multimodal imaging pipelines to understand molecular interactions underpinning disease.

Extending across several schools (Basic & Biomedical Sciences, Cancer & Pharmaceutical, Oral & Craniofacial, Cardiovascular Medicine, Immunology & Microbial) and four faculties at King's, the research proposed to utilize this technology encompasses a wide range of biological applications including cancer, neuroscience, immunology and anatomy. We have selected several projects that span the broad spectrum of research which will be facilitated through the establishment of this system. The strength and depth in our broader user base across King's and across London is considerable and will further strengthen the EuroBioImaging UK Node at King's to enable access from across Europe.

Fluorescence lifetime imaging Microscopy (FLIM) is a technique used to visualise and quantify local variations in the cellular microenvironment. Lifetime image contrast can report pH, viscosity, temperature, refractive index, ion concentrations (i.e. oxygen, calcium) and to extract and quantify important metabolic information from tissue autofluoresence. FLIM, when used with the Förster resonance energy transfer (FRET) technique, enables the quantification of protein-protein or Protein-DNA interactions and conformational changes, which are essential to understanding the complexities of dynamic biological. Despite being recognised as a powerful, highly adaptable, and sensitive tool, the complexity of FLIM technology and analysis procedures coupled with challenges in implementation across the breadth of biomedical sample types mean it has yet to be widely adopted by the bioimaging community. King's College London is a key global hub for advanced fluorescence lifetime imaging in a biological context with both high-level instrument development at the cutting edge for super-resolution, volumetric and high-content imaging and the biological imaging community equipped to exploit and exemplify these platforms. To remain at the forefront of research at the interface between technology development, we propose the Leica Stellaris 8 Dive Falcon platform equipped with a Coherent Discovery NX, offering both confocal and multiphoton excitation, across a broad excitation spectrum combined with multichannel FLIM acquisition. The Falcon represents the current commercial state-of-the art for FLIM, is highly appropriate to support the proposed science and will be highly complementary to cutting-edge developments at King' We aim to democratise the use of FLIM across a far broader base of biological samples and encourage the use of this powerful technique in multimodal imaging pipelines to understand molecular interactions underpinning disease.