Digital holographic microscopy for tracking micro-organisms in 3D

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The majority of single-celled organisms actively navigate their environment. Many species of bacteria, for instance, use a tiny rotary motor to drive a helical filament to generate propulsion. In the case of bacteria, active navigation is required to source nutrients and to avoid toxins, to form symbiotic relationships with each other, and to move towards sites for pathogenic invasion. Currently, the only method we have of studying bacterial swimming is to look at 2D images from a conventional microscope and to infer the 3D nature of the swimming as the bacteria swim in and out of the microscope focus. A full 3D understanding of bacterial swimming is vital to probe many of the unanswered questions about how they move.The aim of this project is to develop a new microscopy technique that is capable of recording the 3D positions of swimming bacteria at high speed. This will be achieved through developing a technique known as digital holographic microscopy. A hologram is an interference pattern between light emanating from a sample and a known reference beam. The fact that we know what this 'reference beam' is a priori means we can process the interference pattern in a computer to generate three-dimensional information about the sample we have imaged. A digital holographic microscope uses a microscope objective to greatly magnify the sample before forming the hologram. In this way we can obtain the 3D positions of organisms as small as bacteria, which are 1-2 microns long.Two recent technological developments allow us to study these microorganisms in 3D, and at high speeds. The first is the advance in digital camera technology. New camera chips now have sufficient pixel resolution to capture the fine detail of these magnified holograms at very fast frame-rates (up to 2,000 frames per second). The second is the availability of graphical processing units (GPUs) for image processing. GPUs were originally invented for the computer gaming industry, but have now found a place in digital processing applications as they are far more efficient at processing large volumes of data than conventional CPUs. Our 3D holographic microscope will necessarily generate huge amounts of data (1GB per frame for a typical 3D hologram); hence, GPUs are integral to the handling of that information. One of the largest stumbling blocks that explains why digital holographic imaging has not yet been used in this manner is the lack of available GPU-compatible software to effectively process these volumes of data.Our proposed technology will bring together microscopy, high-speed, high-resolution digital cameras and the processing power of GPUs to enable the three-dimensional study of bacteria and other microorganisms. It will involve a specific form of holography called 'off-axis holography'. This technique is more difficult to implement optically at higher magnifications, yet it offers improved resolution over 'inline holography' - the other main branch of the technology. Having developed 3D microscopic imaging, we propose two methods in which it will be used to study how bacteria swim. The first uses a relatively low magnification microscope (40x) to capture holograms of many bacteria swimming in one field of view. We will use computer software to track the motion of these bacteria in 3D. In this way, we can investigate the collective swimming behaviour of a population of organisms. The second method uses higher magnification (225x) optics to investigate how individual bacteria interact with their fluid environment. This example would capture the motion of tiny 'tracer' particles that allow us to visualize the fluid flow caused by a single bacterium swimming past. We anticipate that this technology would have a wider appeal to many branches of science interested in how microorganisms and other cells - such as sperm - move.