Biophotonic measurement of forces with subcellular resolution in living, moving tissue
Lead Research Organisation:
University of Glasgow
Department Name: School of Physics and Astronomy
Abstract
At the level of individual cells, forces play a critical role in shaping and regulating tissue architecture and function throughout the lifetime of an organism, from early embryonic development to advanced age. Disruption to this force landscape in ageing, following a heart attack, and in other disease conditions is also recognised to be the root cause of many undesirable changes in tissue structure that ensue. However, these forces have never been measured at a cellular level inside a living animal, in fast-moving tissues such as the beating heart where forces are strong and rapidly fluctuating. A critical issue holding up biomedical research progress is the inability to measure these forces under real-world conditions.
We will develop a new biophotonic approach that will for the first time permit direct imaging and measurement of cell tension forces with subcellular resolution, in rapidly-moving environments such as the cardiovascular system. FLIM-FRET (Fluorescence Lifetime Imaging Microscopy with Förster Resonant Energy Transfer) probes offer the exciting ability to make direct measurements of forces and other properties on nanoscopic scales - which is potentially game-changing for biological research. However, FLIM-FRET is a technically advanced technique to implement even under well-controlled conditions, and the complex tissue motions present within a living animal bring significant further challenges that have not yet been overcome. This is especially true in the dynamic environment of the cardiovascular system. We will therefore develop new computational microscopy techniques to tackle this challenge of measuring forces in moving tissue.
The recent arrival to market of the first widefield time-correlated single-photon counting (TCSPC) array cameras has provided a means for massively parallel FLIM timing measurements of single photons. We will partner this capability with novel motion-correction strategies to develop a unique capability for fast, robust FLIM-FRET in highly dynamic tissues, and achieve world-first measurements of forces with subcellular resolution in the living, beating heart.
Having developed our new approach, we will apply it to two ambitious biological challenge scenarios in heart tissue. First we will make direct measurements of cell-cell and cell-matrix tensions in 2D cultured human-derived cardiomyocytes. This is a model scenario widely used for evaluating new drugs, but clearly one that is hugely simplified compared to the human body. Our force-based investigation of the limitations of this model will ultimately lead to improved drug evaluation techniques, and thus faster and more reliable discovery of new medical treatments.
Having refined our approaches on this scenario, we will then tackle our headline challenge goal of measuring cell-cell tension in the 3D beating zebrafish heart itself, providing a completely new method for biologists to understand the role of tension in driving the heart's normal development and its recovery following injury.
Within the project timeline we are targeting forces in the heart, but we envision broad applications for our approach including: other types of FLIM-FRET probe; other motile environments such as the lungs and gut; measurements in behaving animals. We anticipate that, during and beyond the project lifetime, our approach will unlock biophysical insights into key questions about the role of cellular forces in shaping tissue architecture and function - ultimately impacting understanding and treatment of human heart diseases. Our work will accelerate discovery of new chemical pathways in cells that can be targeted by drugs for the next generation of treatments for human heart diseases.
We will develop a new biophotonic approach that will for the first time permit direct imaging and measurement of cell tension forces with subcellular resolution, in rapidly-moving environments such as the cardiovascular system. FLIM-FRET (Fluorescence Lifetime Imaging Microscopy with Förster Resonant Energy Transfer) probes offer the exciting ability to make direct measurements of forces and other properties on nanoscopic scales - which is potentially game-changing for biological research. However, FLIM-FRET is a technically advanced technique to implement even under well-controlled conditions, and the complex tissue motions present within a living animal bring significant further challenges that have not yet been overcome. This is especially true in the dynamic environment of the cardiovascular system. We will therefore develop new computational microscopy techniques to tackle this challenge of measuring forces in moving tissue.
The recent arrival to market of the first widefield time-correlated single-photon counting (TCSPC) array cameras has provided a means for massively parallel FLIM timing measurements of single photons. We will partner this capability with novel motion-correction strategies to develop a unique capability for fast, robust FLIM-FRET in highly dynamic tissues, and achieve world-first measurements of forces with subcellular resolution in the living, beating heart.
Having developed our new approach, we will apply it to two ambitious biological challenge scenarios in heart tissue. First we will make direct measurements of cell-cell and cell-matrix tensions in 2D cultured human-derived cardiomyocytes. This is a model scenario widely used for evaluating new drugs, but clearly one that is hugely simplified compared to the human body. Our force-based investigation of the limitations of this model will ultimately lead to improved drug evaluation techniques, and thus faster and more reliable discovery of new medical treatments.
Having refined our approaches on this scenario, we will then tackle our headline challenge goal of measuring cell-cell tension in the 3D beating zebrafish heart itself, providing a completely new method for biologists to understand the role of tension in driving the heart's normal development and its recovery following injury.
Within the project timeline we are targeting forces in the heart, but we envision broad applications for our approach including: other types of FLIM-FRET probe; other motile environments such as the lungs and gut; measurements in behaving animals. We anticipate that, during and beyond the project lifetime, our approach will unlock biophysical insights into key questions about the role of cellular forces in shaping tissue architecture and function - ultimately impacting understanding and treatment of human heart diseases. Our work will accelerate discovery of new chemical pathways in cells that can be targeted by drugs for the next generation of treatments for human heart diseases.