Periodic Reporting for period 5 - TransPhorm (Single molecule imaging of transmembrane protein structure and function in their native state)
Periodo di rendicontazione: 2021-09-01 al 2022-11-30
TransPhorm will pioneer a transformative technology that will enable the study of transmembrane protein (TMP) structure and function in their natural state with unprecedented sensitivity and resolution. TMPs play a crucial role in cell signalling and are essential for life.
The overarching ambition of TransPhorm is to establish a revolutionary technology to deliver structural, functional and localisation information with unprecedented sensitivity and resolution of TMPs in their native environment. This will deliver tremendous scientific gains and more broadly impact positively on the health and wellbeing of society.
The primary objectives are:
1. Obtain high-resolution structural information of TMPs in their natural state by developing a quantum sensing protocol using Nitrogen Vacancy defects in diamond.
2. Study ion channel function with high temporal resolution and ion selectivity by uniquely employing NV nanodiamonds as magnetic field sensors in solution state nuclear magnetic resonance.
3. Track the location of ion channels in the plasma membrane over time with video rate, multi-modal super resolution optical microscopy.
4. Determine the structure, function and location of ion channels in their natural state through exemplar studies using model TMPs, native TMPs and live cells.
The overarching ambition of TransPhorm is to establish a revolutionary technology to deliver structural, functional and localisation information with unprecedented sensitivity and resolution of TMPs in their native environment. This will deliver tremendous scientific gains and more broadly impact positively on the health and wellbeing of society.
The primary objectives are:
1. Obtain high-resolution structural information of TMPs in their natural state by developing a quantum sensing protocol using Nitrogen Vacancy defects in diamond.
2. Study ion channel function with high temporal resolution and ion selectivity by uniquely employing NV nanodiamonds as magnetic field sensors in solution state nuclear magnetic resonance.
3. Track the location of ion channels in the plasma membrane over time with video rate, multi-modal super resolution optical microscopy.
4. Determine the structure, function and location of ion channels in their natural state through exemplar studies using model TMPs, native TMPs and live cells.
Main results of this work include, integration of Nitrogen Vacancy (NV) rich nanoscale diamond sensors into electronspun polymer nanofibers to replicate the physiological niche, particularly important for neuronal and stem cell culture. The electrospun fibre diamond sensing platform supported the growth of neural stem cells and their differentiated progeny and enabled collection of ODMR signals is a physiologically relevant niche. Experimental studies investigating cells cultured on planar diamond chips were also performed. The emphasis of this work was detection of paramagnetic contrast agents used in conventional MRI. NV sensing protocols were applied to image the extracellular pooling of contrast agents and internalisation of contrast agents in cells.
Sensing protocols were designed and exemplar studies performed to study cellular respiratory processes (live cells and mitochondrial extracts), discrimination of diseased cells from healthy cells based on detection of spin active species (Wild type Alzheimer’s cells vs Alzheimer’s cells with mutations), validation of detection of
hydroxyl radicals from cells (NV sensing protocols vs Electron Paramagnetic Resonance (EPR)) and super resolution imaging of nanodiamonds conjugated with antibodies for specific cell membrane proteins. Further studies demonstrated the successful integration of plannar diamond chips with functioning neuronal networks, as verified by conventional calcium reporters.
Instrumentation for optical detection of magnetic resonance and localisation of transmembrane receptors was developed. Here a new approach to localisation microscopy based on the intrinsic blinking of NV photoluminescence in a narrow wavelength emission band in physiologically relevant buffers used developed. Assessment of TMPs in fixed cells and tissue has been carried out using this imaging technique. Specifically, tissue studies were performed to attain single molecule localisation of ryanodine receptors in rat skeletal muscle sections. Additionally, cell studies enabled imaging of nuclear pore complexes, the multiprotein channels that connect the nucleus and cytoplasm of eukaryotic cells.
A new experimental system was developed enabling correlative optically detected magnetic resonance (ODMR) studies with electron microscopy. This approach involves drop casting nanodiamonds on a transmission electron microscope (TEM) finder grid such that samples of interest can be evaluated both at an atomic scale via TEM and their functional and paramagnetic profile assessed via NV based sensing. Several examplar studies were performed that demonstrated the ability to detect free radicals from nanoscale assemblies that will translate well for the study of sub-cellular components and transmembrane proteins.
Sensing protocols were designed and exemplar studies performed to study cellular respiratory processes (live cells and mitochondrial extracts), discrimination of diseased cells from healthy cells based on detection of spin active species (Wild type Alzheimer’s cells vs Alzheimer’s cells with mutations), validation of detection of
hydroxyl radicals from cells (NV sensing protocols vs Electron Paramagnetic Resonance (EPR)) and super resolution imaging of nanodiamonds conjugated with antibodies for specific cell membrane proteins. Further studies demonstrated the successful integration of plannar diamond chips with functioning neuronal networks, as verified by conventional calcium reporters.
Instrumentation for optical detection of magnetic resonance and localisation of transmembrane receptors was developed. Here a new approach to localisation microscopy based on the intrinsic blinking of NV photoluminescence in a narrow wavelength emission band in physiologically relevant buffers used developed. Assessment of TMPs in fixed cells and tissue has been carried out using this imaging technique. Specifically, tissue studies were performed to attain single molecule localisation of ryanodine receptors in rat skeletal muscle sections. Additionally, cell studies enabled imaging of nuclear pore complexes, the multiprotein channels that connect the nucleus and cytoplasm of eukaryotic cells.
A new experimental system was developed enabling correlative optically detected magnetic resonance (ODMR) studies with electron microscopy. This approach involves drop casting nanodiamonds on a transmission electron microscope (TEM) finder grid such that samples of interest can be evaluated both at an atomic scale via TEM and their functional and paramagnetic profile assessed via NV based sensing. Several examplar studies were performed that demonstrated the ability to detect free radicals from nanoscale assemblies that will translate well for the study of sub-cellular components and transmembrane proteins.
This work conceived and demonstrated novel methodologies to support NV based sensing in biological systems. A previously unreported quantum-sensing platform that mimics the nanoscale architecture and topography of the cell niche has been developed. This sensing platform enabled acquisition of ODMR spectra, longitudinal spin relaxometry measurements and external magnetic field detection. The work advances the current state of the art in quantum sensing by providing a versatile sensing platform that recapitulates physiological-like cell niches and provides fast measurement protocols for detection of co-ordinated endogenous signals from clinically relevant populations of electrically active neuronal circuits.
A further advance is the establishment of a new optical sensing protocol with sub-second time response for monitoring paramagnetic species. The protocol enabled detection of changes in the concentration of paramagnetic ions in proximity to a planar diamond chip containing near surface ensembles of NVs. The established protocol operates at room temperature with small sample requirements and a relatively straightforward experimental protocol and data acquisition and analysis. The sub-second time resolution will allow the monitoring of endogenous paramagnetic species production on timescales relevant to discrete signalling pathways related to onset of disease with the scope of aiding therapeutic strategies.
A significant development was the establishment of a novel imaging protocol that used the intrinsically-activated photochromism of nanoscale diamond underr laser illumination intensities of the order of 100-times lower than that required for conventional localisation microscopy (i.e. dSTORM). Exemplar studies localising transmembrane channel receptors in skeletal muscle cells produced super resolution images 3 to-5 fold faster compared to dSTORM with lateral spatial resolution of the order of 20 nm.
A new protocol for integration of NV diamond sensors in a spontaneously active network of neuronal cells, of relevance to the field of neuroscience, was developed. Photoluminescence from NV centers was detected with high spatiotemporal resolution using measurement protocols potentially capable of single shot detection of neuronal activity. Spatially correlated images of photoluminescence from NV centers were recorded and validated by calcium probes demonstrating successful acquisition of optically detected magnetic resonance from regions of endogenously firing neuronal cultures.
A method for mitochondrial free radical detection via modulation of photoluminescence from nitrogen vacancies in diamond was developed. This method enables the study the of the whole process of oxidative phosphorylation and spin active intermediaries of biological processes with potential to elucidate the biophysical parameters underpinning mitochondrial function and dysfunction.
Workflows combining the power of two local probes, namely, Nitrogen Vacancy (NV) spin active defects in diamond and an electron beam, within experimental platforms used in electron microscopy were established. The measurement strategies demonstrated provide a pathway for quantitative quantum sensing with atomic-scale spatial resolution, critical to the development of quantum technologies and single molecules studies in biology.
A further advance is the establishment of a new optical sensing protocol with sub-second time response for monitoring paramagnetic species. The protocol enabled detection of changes in the concentration of paramagnetic ions in proximity to a planar diamond chip containing near surface ensembles of NVs. The established protocol operates at room temperature with small sample requirements and a relatively straightforward experimental protocol and data acquisition and analysis. The sub-second time resolution will allow the monitoring of endogenous paramagnetic species production on timescales relevant to discrete signalling pathways related to onset of disease with the scope of aiding therapeutic strategies.
A significant development was the establishment of a novel imaging protocol that used the intrinsically-activated photochromism of nanoscale diamond underr laser illumination intensities of the order of 100-times lower than that required for conventional localisation microscopy (i.e. dSTORM). Exemplar studies localising transmembrane channel receptors in skeletal muscle cells produced super resolution images 3 to-5 fold faster compared to dSTORM with lateral spatial resolution of the order of 20 nm.
A new protocol for integration of NV diamond sensors in a spontaneously active network of neuronal cells, of relevance to the field of neuroscience, was developed. Photoluminescence from NV centers was detected with high spatiotemporal resolution using measurement protocols potentially capable of single shot detection of neuronal activity. Spatially correlated images of photoluminescence from NV centers were recorded and validated by calcium probes demonstrating successful acquisition of optically detected magnetic resonance from regions of endogenously firing neuronal cultures.
A method for mitochondrial free radical detection via modulation of photoluminescence from nitrogen vacancies in diamond was developed. This method enables the study the of the whole process of oxidative phosphorylation and spin active intermediaries of biological processes with potential to elucidate the biophysical parameters underpinning mitochondrial function and dysfunction.
Workflows combining the power of two local probes, namely, Nitrogen Vacancy (NV) spin active defects in diamond and an electron beam, within experimental platforms used in electron microscopy were established. The measurement strategies demonstrated provide a pathway for quantitative quantum sensing with atomic-scale spatial resolution, critical to the development of quantum technologies and single molecules studies in biology.