BioAFM: Integrated atomic force and light microscopy for mechanochemical cell biology
Lead Research Organisation:
University of Warwick
Department Name: Warwick Medical School
Abstract
We explore the environment surrounding us through the combination of multiple senses, i.e., sight, hearing, smell, taste, and touch. Sensing with light is very powerful as it allows us to detect objects without direct contact. However, there are types of information that can't be obtained by sight alone. For example, touch tells us the mechanical properties of an object. By touching someone's hand with your finger, you can feel the texture of the skin and how soft or hard the skin is. You can also find how many fingers exist on that hand and even the bones beneath the skin and flesh.
Life science at the scale of molecular machinery, cells and tissues has been heavily relying on light microscopy especially when we try to observe them in their living state. Measurement of the mechanical properties by 'touching' should add another dimension to our sensing of the biological object and provides information that can't be obtained by light microscopy. An atomic force microscope (AFM) is an instrument that achieves such measurements.
We integrate an AFM with a light microscope that has two different modes of observation, 1) total internal fluorescence microscopy (TIRFM), which can detect single molecules by fluorescence, and 2) high-resolution 3D fluorescence microscopy (3DFM), which is suitable for observation of thick samples such as cell and tissues. The combination of AFM and TIRFM will allow us to visualize the live dynamics of the assembly of biomolecular machines that play key roles in the transport/traffic inside the cell. AFM+3DFM enables us to measure various mechanical properties of a single cell of interest in a complex multicellular environment by using a fluorescent marker as a guide.
Our proposed and future BioAFM experiments will give new insights into cellular and molecular mechanisms of cell and tissue morphogenesis and other biological processes, in which mechanical force plays a key role.
Life science at the scale of molecular machinery, cells and tissues has been heavily relying on light microscopy especially when we try to observe them in their living state. Measurement of the mechanical properties by 'touching' should add another dimension to our sensing of the biological object and provides information that can't be obtained by light microscopy. An atomic force microscope (AFM) is an instrument that achieves such measurements.
We integrate an AFM with a light microscope that has two different modes of observation, 1) total internal fluorescence microscopy (TIRFM), which can detect single molecules by fluorescence, and 2) high-resolution 3D fluorescence microscopy (3DFM), which is suitable for observation of thick samples such as cell and tissues. The combination of AFM and TIRFM will allow us to visualize the live dynamics of the assembly of biomolecular machines that play key roles in the transport/traffic inside the cell. AFM+3DFM enables us to measure various mechanical properties of a single cell of interest in a complex multicellular environment by using a fluorescent marker as a guide.
Our proposed and future BioAFM experiments will give new insights into cellular and molecular mechanisms of cell and tissue morphogenesis and other biological processes, in which mechanical force plays a key role.
Technical Summary
Life science at the scale of cells and tissues has been heavily reliant on light microscopy. Orthogonal information such as the measurement of mechanical properties would add another dimension to our understanding of the mechanisms of life. An atomic force microscope (AFM) is an instrument that achieves such measurements. A recent model of AFM is compatible with light microscopes. It offers various modes of nanomechanical measurements such as peak force quantitative imaging (PeakForce-QI), nano-manipulation and -indentation, and single-cell force spectroscopy.
This facilitates two classes of experiments
1. Observation of dynamics of biological molecules and their higher-order assemblies at high spatial (~nm) and temporal (~s) resolutions.
2. Measurements of mechanical properties of molecular assemblies, cells, and tissues.
In the BioAFM platform we request, we will probe biological samples at molecular resolution by coupling the AFM with total internal reflection microscopy (TIRFM) to detect single fluorescently tagged molecules. This will allow us to observe the dynamic process of macromolecular assembly, such as microtubules and clathrin cages, at high spatial (~nm) and temporal (~) precision.
By combination of AFM with 3D fluorescence microscopy, we will be able to correlate the dynamic behaviour of the cytoskeletal proteins with the mechanical properties of the cell. By using a fluorescence marker as a guide, we will be able to pinpoint a cell within a complex tissue environment and measure its mechanical properties through various modalities of the AFM measurement.
By this way, BioAFM will bring us to a novel understanding of the cellular and molecular mechanisms of complex morphogenesis, in which mechanical forces play key roles.
This facilitates two classes of experiments
1. Observation of dynamics of biological molecules and their higher-order assemblies at high spatial (~nm) and temporal (~s) resolutions.
2. Measurements of mechanical properties of molecular assemblies, cells, and tissues.
In the BioAFM platform we request, we will probe biological samples at molecular resolution by coupling the AFM with total internal reflection microscopy (TIRFM) to detect single fluorescently tagged molecules. This will allow us to observe the dynamic process of macromolecular assembly, such as microtubules and clathrin cages, at high spatial (~nm) and temporal (~) precision.
By combination of AFM with 3D fluorescence microscopy, we will be able to correlate the dynamic behaviour of the cytoskeletal proteins with the mechanical properties of the cell. By using a fluorescence marker as a guide, we will be able to pinpoint a cell within a complex tissue environment and measure its mechanical properties through various modalities of the AFM measurement.
By this way, BioAFM will bring us to a novel understanding of the cellular and molecular mechanisms of complex morphogenesis, in which mechanical forces play key roles.
