Quantitative phase imaging microscopy for label-free, high-content imaging
Lead Research Organisation:
University of Warwick
Department Name: Warwick Medical School
Abstract
Optical microscopy is a routine tool in biomedical research, allowing us to view the inner workings of cells and how cells work together to form tissues. Being able to see the movement of cells helps us to understand important processes in healthy cells and tissues and what goes wrong in disease.
Cells are inherently transparent and difficult to image in their native state. Typically, we add fluorescent markers to enable visualisation of cellular substructures or specific proteins. While such fluorescent probes result in excellent high contrast images, both the introduction and imaging of the probe can be detrimental to cell viability and disturb the observed process. The binding of a bulky fluorescent molecule to a protein of interest, for example, can affect the behaviour of that molecule, while fluorescence imaging produces free radicals which are harmful to the cells. In some cells, such as patient-derived cells or other sensitive cell types, it may not be possible to add fluorescent markers at all, making fluorescence imaging impossible.
Light transmitted through transparent cells undergoes a phase shift, by interfering this light with a reference beam, we can generate holograms. Spatial information in the form of cellular density can be extracted from these holograms using carefully designed algorithms. The images generated from these holograms resemble those achieved from fluorescently labelled cells, supplying high contrast images without requiring fluorescent markers. This technique, known as quantitative phase imaging, allows us to image cells in their native state with minimal perturbation.
This application requests funds to purchase a quantitative phase imaging microscope for minimally invasive cellular imaging. The requested microscope will come with fully automated acquisition and analysis, enabling us to image up to 96 samples in parallel and extract information on the proliferation, movement and shape of cells automatically in an objective manner. This microscope will therefore support cell-based screening for a range of discovery and translational research projects to understand and develop treatments for rare genetic diseases, bacterial infections and cancer.
Cells are inherently transparent and difficult to image in their native state. Typically, we add fluorescent markers to enable visualisation of cellular substructures or specific proteins. While such fluorescent probes result in excellent high contrast images, both the introduction and imaging of the probe can be detrimental to cell viability and disturb the observed process. The binding of a bulky fluorescent molecule to a protein of interest, for example, can affect the behaviour of that molecule, while fluorescence imaging produces free radicals which are harmful to the cells. In some cells, such as patient-derived cells or other sensitive cell types, it may not be possible to add fluorescent markers at all, making fluorescence imaging impossible.
Light transmitted through transparent cells undergoes a phase shift, by interfering this light with a reference beam, we can generate holograms. Spatial information in the form of cellular density can be extracted from these holograms using carefully designed algorithms. The images generated from these holograms resemble those achieved from fluorescently labelled cells, supplying high contrast images without requiring fluorescent markers. This technique, known as quantitative phase imaging, allows us to image cells in their native state with minimal perturbation.
This application requests funds to purchase a quantitative phase imaging microscope for minimally invasive cellular imaging. The requested microscope will come with fully automated acquisition and analysis, enabling us to image up to 96 samples in parallel and extract information on the proliferation, movement and shape of cells automatically in an objective manner. This microscope will therefore support cell-based screening for a range of discovery and translational research projects to understand and develop treatments for rare genetic diseases, bacterial infections and cancer.
Technical Summary
This proposal is to request funds to purchase a high-content quantitative phase imaging (QPI) microscope. This microscope will allow label-free imaging of cells with minimal phototoxicity and artefacts. The resulting high contrast images allow automatic segmentation and tracking of cells. Automated acquisition and analysis of QPI images from multi-well plates enable long-term recording of cell migration and proliferation of even the most sensitive cells. Parameters that will be measured over timeframes of up to several days include mitotic timing, speed and directionality of cell migration, dry mass increase and cell morphology changes. These assays therefore allow (i) the careful validation of clonal cell lines for discovery projects, (ii) cell-based drug discovery screens, (iii) monitoring circadian oscillations in cell behaviour, (iv) investigating the uncoupling of cell growth and division of bacteria with antibiotic treatments, (v) optimise phage therapy in an in vitro model. We propose 11 research projects that will benefit from access to a QPI system at Warwick addressing challenges in precision medicine, advanced therapies and antimicrobial resistance.
The system will be managed and made available within Warwick's Computing and Advanced Microscopy Development Unit (CAMDU). CAMDU is a team of imaging specialists and a computational image analysis expert who manage access, user support, maintenance and assistance with data storage and analysis from 20 advanced light microscopes. These systems all focus on high-resolution fluorescence imaging and the requested QPI would complement this portfolio with a high-content label-free imaging modality. CAMDU have extensive experience in effectively managing high-end multiuse microscopy equipment and making it available to even unexperienced users. An example is our Lattice Light Sheet visitor programme.
The system will be managed and made available within Warwick's Computing and Advanced Microscopy Development Unit (CAMDU). CAMDU is a team of imaging specialists and a computational image analysis expert who manage access, user support, maintenance and assistance with data storage and analysis from 20 advanced light microscopes. These systems all focus on high-resolution fluorescence imaging and the requested QPI would complement this portfolio with a high-content label-free imaging modality. CAMDU have extensive experience in effectively managing high-end multiuse microscopy equipment and making it available to even unexperienced users. An example is our Lattice Light Sheet visitor programme.
