Single-cell nanobiopsy for spatial and temporal profiling of living cells
Author(s)
Sahota, Annpreet
Type
Thesis
Abstract
Single-cell analysis is a rapidly growing field that is reshaping our understanding of fundamental biological processes and complex diseases. Subcellular sampling techniques advance from previous single-cell methods as they maintain cells in their standard microenvironment and do not require cell lysis or fixation. These have the potential to profile the single-cell omics of living cells and aid our understanding of intracellular mechanisms. However, many existing subcellular sampling tools involve the aspiration of cytoplasmic fluid, raising questions regarding the viability of cells with repeat sampling. In this work, minimally invasive dielectrophoretic nanoscale tweezers (nanotweezers) are employed for precise sampling of RNA and single mitochondrion in living neurons. With this nanobiopsy tool, biomolecules are trapped directly from cells, avoiding the need to remove large cytoplasmic volumes.
The nanotweezer platform is developed to provide spatial and temporal measurements of biomolecules in live single cells. Following optimising the dielectrophoretic trapping capabilities of the nanotweezer, we show that mRNA can be successfully isolated from different compartments of live rat hippocampal neurons with high spatial resolution. Repeat sampling from the same cell with minimal impact on cell viability and the detection of RNAs from individual nanobiopsies is achieved. As a result, the tool is used to study dendritic localisation of mRNAs, including measuring dynamic changes in gene expression in the same living cell.
Single mitochondrion extraction from the neurites of single neurons is also presented, alongside developing methods for single mitochondrion analysis. Finally, the effects of α-synuclein on single-cell mitochondrial expression are investigated, where we demonstrate the removal of mitochondria from the same living neuron before and after treatment. This work shows promising results for future expansion of the tool for dynamic and precise spatial quantification of biomolecules in single cells which could ultimately have significant implications in spatial transcriptomics, deciphering cellular mechanics, and tracking disease.
The nanotweezer platform is developed to provide spatial and temporal measurements of biomolecules in live single cells. Following optimising the dielectrophoretic trapping capabilities of the nanotweezer, we show that mRNA can be successfully isolated from different compartments of live rat hippocampal neurons with high spatial resolution. Repeat sampling from the same cell with minimal impact on cell viability and the detection of RNAs from individual nanobiopsies is achieved. As a result, the tool is used to study dendritic localisation of mRNAs, including measuring dynamic changes in gene expression in the same living cell.
Single mitochondrion extraction from the neurites of single neurons is also presented, alongside developing methods for single mitochondrion analysis. Finally, the effects of α-synuclein on single-cell mitochondrial expression are investigated, where we demonstrate the removal of mitochondria from the same living neuron before and after treatment. This work shows promising results for future expansion of the tool for dynamic and precise spatial quantification of biomolecules in single cells which could ultimately have significant implications in spatial transcriptomics, deciphering cellular mechanics, and tracking disease.
Version
Open Access
Date Issued
2024-01-17
Date Awarded
2024-03-01
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Ivanov, Aleksandar
Edel, Joshua
Devine, Michael
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)