Confocal image acquisition system with capacity for robotic fluid additions: flexible tool for high-content screening
Lead Research Organisation:
University College London
Department Name: Neuroscience Physiology and Pharmacology
Abstract
Experiments investigating cell physiology and drug responses often exploit light-emitting (fluorescent) proteins and probes. Fluorescent probes, engineered to report on different cellular parameters, can be used as "biosensors". In the past, these experiments were conducted examining a small visual field under a microscope, manually adding compounds to dishes carrying cells, and selecting a small number of cells on which to measure changes. New image-acquisition systems allow this process to be done in an automated way. Cells are grown on plates with tens or hundreds of separate wells. At specified time points in the experiment, robotic arms add different compounds to the different wells, while images are captured by a camera which detects light of different colours, emitted by different biosensors. Small, localised changes in response to perturbations are captured rapidly and continuously over a period of time. Automated computer-based analysis of the images greatly increases the efficiency of work.
Such image-acquisition systems have made it possible to increase the number of cells studied by orders of magnitude. This has eliminated experimenter bias and allowed us to focus on subsets of cells. Optical resolution has improved, so we can even study sub-cellular structures, called organelles. In addition, the possibility of using multiple biosensors simultaneously has allowed measurement of multiple characteristics in the same cell/organelle. Assays with multiple readouts are described as having "high-content". Furthermore many different conditions can be rapidly compared on a single plate, (e.g. composition of the fluid added, genetic makeup of cells, etc.). This allows rapid "screening" of a large number of compounds, for instance, which can be useful when developing drugs.
The image-acquisition system we propose to buy will be used by many research groups, and will be available across the UCL campus. It will allow us to develop and run different high-content assays.
One proposed study will explore potential new therapies for cancer. All our cells have mitochondria, organelles that burn fuels and generate packages of energy, readily available for the cell's needs. Mitochondria have their own genetic material, mtDNA, distinct from that in the cell's nucleus. Scientists have discovered that tumour cells very often have changes, called mutations, in their mtDNA, not present in the surrounding healthy tissues. These mtDNA mutations can be used as targeting labels, directing specialized enzymes (called mitoTALENs) to make damaging cuts in mtDNA from tumour cells, without affecting the nearby tissues. Cells with damaged mtDNA grow more slowly, and are more susceptible to chemotherapy drugs. Using high-content screening techniques, tumour and healthy cells will be treated with mitoTALENs under a variety of different conditions. The information gained will validate the approach, and lay foundations for future therapy development.
Another lab will work on improving treatment for people with cystic fibrosis (CF). In CF the CFTR protein is missing or defective. CFTR regulates flow of anions (negatively charged chloride and bicarbonate ions) into and out of cells that line ducts of our body (airways, intestine, pancreas, liver etc). The flow of bicarbonate is especially important for controlling mucous secretions produced by these duct cells. CFTR-targeted drugs can help CF patients, but we know that, at least in liver ducts, current drugs restore chloride but not bicarbonate flow. We will generate a model anion flux biosensor system. This will allow us to rapidly monitor chloride and bicarbonate flow and to determine how CFTR drugs affect it for 62 different variants of CFTR found in patients. What we will learn about processes at the root of CF disease will help clinicians choose the best drugs for individual patients, and guide future drug development.
Such image-acquisition systems have made it possible to increase the number of cells studied by orders of magnitude. This has eliminated experimenter bias and allowed us to focus on subsets of cells. Optical resolution has improved, so we can even study sub-cellular structures, called organelles. In addition, the possibility of using multiple biosensors simultaneously has allowed measurement of multiple characteristics in the same cell/organelle. Assays with multiple readouts are described as having "high-content". Furthermore many different conditions can be rapidly compared on a single plate, (e.g. composition of the fluid added, genetic makeup of cells, etc.). This allows rapid "screening" of a large number of compounds, for instance, which can be useful when developing drugs.
The image-acquisition system we propose to buy will be used by many research groups, and will be available across the UCL campus. It will allow us to develop and run different high-content assays.
One proposed study will explore potential new therapies for cancer. All our cells have mitochondria, organelles that burn fuels and generate packages of energy, readily available for the cell's needs. Mitochondria have their own genetic material, mtDNA, distinct from that in the cell's nucleus. Scientists have discovered that tumour cells very often have changes, called mutations, in their mtDNA, not present in the surrounding healthy tissues. These mtDNA mutations can be used as targeting labels, directing specialized enzymes (called mitoTALENs) to make damaging cuts in mtDNA from tumour cells, without affecting the nearby tissues. Cells with damaged mtDNA grow more slowly, and are more susceptible to chemotherapy drugs. Using high-content screening techniques, tumour and healthy cells will be treated with mitoTALENs under a variety of different conditions. The information gained will validate the approach, and lay foundations for future therapy development.
Another lab will work on improving treatment for people with cystic fibrosis (CF). In CF the CFTR protein is missing or defective. CFTR regulates flow of anions (negatively charged chloride and bicarbonate ions) into and out of cells that line ducts of our body (airways, intestine, pancreas, liver etc). The flow of bicarbonate is especially important for controlling mucous secretions produced by these duct cells. CFTR-targeted drugs can help CF patients, but we know that, at least in liver ducts, current drugs restore chloride but not bicarbonate flow. We will generate a model anion flux biosensor system. This will allow us to rapidly monitor chloride and bicarbonate flow and to determine how CFTR drugs affect it for 62 different variants of CFTR found in patients. What we will learn about processes at the root of CF disease will help clinicians choose the best drugs for individual patients, and guide future drug development.
Technical Summary
We request funding for a confocal microscope system capable of automated image aquisition from a variety of multi-well formats, with fluidics. It will allow experimental work to gain insight in biological mechanisms and advance early drug discovery by harnessing automation in image-acquisition technology and quantitative image analysis .
Uniquely, this system allows multiple automated fluid additions, with individual wells receiving distinct solutions. Experiments involving microscopic observation of limited visual fields and repeated manual pipetting can be replaced by semi-automated assays. This will allow (i) a 10-100 fold increase in the number of cells sampled; (ii) enhanced reproducibility and elimination of operator selection bias; (iii) miniaturization, thus lower financial costs and environmental impact. This also enables medium-throughput screening of compounds, useful for early drug discovery investigation of mechanism of action.
The system we propose to purchase includes 7 laser light-sources. The number of available optical probes capable of reporting on cellular parameters, is rapidly expanding, providing valuable tools for investigation. Changes in response to perturbations (in our case, mainly compound additions) can be captured on different wavelength channels with high spatio-temporal resolution. Such "high-content" readouts (multiple dimensions of information on individual cells/organelles), provide synergistic insight on mechanisms underlying those responses.
Several labs at UCL work with such high-content, automated assays. In one lab, an assay that simultaneously quantifies CFTR cellular localization and ion-channel function is being used to develop novel pharmacotherapies for cystic fibrosis and secretory diarrhoeas. In another lab, cancer cell-lines are used for combinatorial (metabolic state, oxygenation, mitochondrial content, chemotherapeutic agent) mapping to investigate new gene-editing treatments targeting mitochondrial DNA.
Uniquely, this system allows multiple automated fluid additions, with individual wells receiving distinct solutions. Experiments involving microscopic observation of limited visual fields and repeated manual pipetting can be replaced by semi-automated assays. This will allow (i) a 10-100 fold increase in the number of cells sampled; (ii) enhanced reproducibility and elimination of operator selection bias; (iii) miniaturization, thus lower financial costs and environmental impact. This also enables medium-throughput screening of compounds, useful for early drug discovery investigation of mechanism of action.
The system we propose to purchase includes 7 laser light-sources. The number of available optical probes capable of reporting on cellular parameters, is rapidly expanding, providing valuable tools for investigation. Changes in response to perturbations (in our case, mainly compound additions) can be captured on different wavelength channels with high spatio-temporal resolution. Such "high-content" readouts (multiple dimensions of information on individual cells/organelles), provide synergistic insight on mechanisms underlying those responses.
Several labs at UCL work with such high-content, automated assays. In one lab, an assay that simultaneously quantifies CFTR cellular localization and ion-channel function is being used to develop novel pharmacotherapies for cystic fibrosis and secretory diarrhoeas. In another lab, cancer cell-lines are used for combinatorial (metabolic state, oxygenation, mitochondrial content, chemotherapeutic agent) mapping to investigate new gene-editing treatments targeting mitochondrial DNA.
