Awarded

Confocal imaging system.

Descriptions

The Francis Crick Institute (‘The Crick’) is a biomedical research institute dedicated to understanding the scientific mechanisms of living things. Its work is to understand why disease develops and to find new ways to treat, diagnose and prevent illnesses such as cancer, heart disease, stroke, infections and neuro-degenerative diseases.The Francis Crick Institute requires full time access to a Confocal Microscope to continue to perform a project requiring long-term live imaging of the Drosophila pupal abdomen. Due to the length of the imaging time period (18 to 20 hours) and the nature of the fluorophores used, the microscope will need to provide the following:— High-sensitivity Gallium Arsenide Phosphide (GaAsP) detectors. To ensure minimal photo-toxicity from the microscope's lasers so that the animal develops normally while still producing clear images, the microscope needs to be equipped with the latest high sensitivity GaAsP detectors. The ability to undertake a future upgrade to optically increase the resolution in X, Y and Z to near super resolution levels, whilst improving the signal to noise ratio for weakly fluorescing live cell imaging is also highly desirable in order to live-image fine actin and adhesion structures during abdominal development.— Reliable optical sectioning to ensure separation of closely packed objects in the Z plane. The Drosophila abdomen is a large tissue that cannot be imaged in a single frame using a 40x objective. Therefore we need the software necessary to seamlessly tile images acquired sequentially on the same animal in order to capture all the information. Furthermore, because of the 3D nature of the tissue and the fact that it is surrounded by other bright objects (muscle, macrophages, soft cuticle etc.,), a point-scanning confocal system is desirable over a spinning disc system in order to separate the abdominal cells away from surrounding cells during post-processing. Our past work with spinning-disc systems indicates these are not suitable for this project as we cannot obtain sufficient separation of the abdominal cells for accurate segmentation.— Even dwell time across the imaged area to ensure reliable quantitative analysis. The quality of the image needs to be such that we can use a series of custom-built computer programmes to recognise and track the behaviour of thousands of individual cells through time. The data generated will then be fed into a vertex-based computational model of the abdomen. In order to ensure reliable quantitative analysis of the images, the scanning mirrors of the microscope need to cross the field of imaging in a linear manner, so that each pixel receives the same dwell time regardless of position in the field of view.— Advanced spectral unmixing using a 32 element GaAsP array with high quantum efficiency allowing high-speed simultaneous imaging of up to 10 fluorophores. The project involves simultaneous acquisition of multiple fluorescently tagged proteins, in particular CFP/YFP/RFP (or similar combinations). As many of these are encoded in transgenic animal controlled by endogenous or weak promoters, fast (4 frames per second or faster at 512x512 resolution), reliable and efficient spectral unmixing is of paramount importance in order to maximise signal across the spectrum, while minimising the possibility of cross-channel interference with other expressed fluorescent proteins of with autofluorescence. We therefore require a spectral GaAsP array with a Quantum Efficiency of at least 45 % and even sensitivity across all elements regardless of spectral positioning. This will ensure that weaker signals are captured with high signal-to-noise ratio and guarantee the absence of spectral bias.— Environmental chamber with both heating and cooling capacity. In order to take advantage of Drosophila genetic tools such as the tubulin-GAL80ts system, we require an environmental chamber with both heating and cooling capacity to reliably maintain temperatures between 18°C and 32°C.