Breakthrough in fast tomographic imaging takes traditional microscopy to a new level
3D imaging has become something of a necessary tool, especially for research which benefits from the dynamic imaging of in-vivo biological processes. To meet this demand biology and preclinical laboratories have drawn on tomographical innovations such as Fluorescence Molecular Tomography (FMT) for imaging in highly scattering media, and Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) for imaging in low scattering specimens. However, while effective, these tools lack throughput or the ability to process samples simultaneously. The EU-funded Marie Curie Career Integration Grant (CIG) supported the HIGH THROUGHPUT TOMO project to address this bottleneck by enabling the creation of a set of tools that achieved very fast optical imaging and data analysis. As well as offering a range of solutions, the project, coordinated by Prof. Manuel Desco and hosted by the Universidad Carlos III de Madrid, concentrated especially on the development of fast tomography in microscopy. Achieving 3D time-lapse imaging of life – as it unfolds Traditional light sheet imaging based on the scanning approach uses motors to move samples, which proves extremely time consuming. Processing 200 images for example, can take 200 seconds. Summarising the key result of HIGH THROUGHPUT TOMO, Marie Curie CIG fellow Dr. Jorge Ripoll says, ‘We optimised the hardware and software arrangement to reduce the need for motor-based movement, replacing it with light beam scanning and tunable lenses with fast response - in the order of 10ms. This reduced the time for each scan by a factor of 100, requiring 2 seconds to acquire the same 200 images instead of 200 seconds.’ At the moment, practically all in-vivo imaging measurements using model organisms such as the zebrafish or the fruit fly, are based on single specimen imaging. This has severe limitations as Dr. Ripoll explains, ‘To obtain scientifically sound results we had to repeat these measurements on large species populations, thus multiplying the time needed for a single specimen by the number of measurements needed. This is not only extremely time-consuming, but maintaining the same conditions is problematic, often resulting in the discarding of measurements.’ The team’s breakthrough, along with some key geometrical changes in the optical imaging setup, means that researchers are now capable of imaging several specimens at the same time. Dr. Ripoll cites the team’s successful imaging in-vivo of the development of 13 fruit flies simultaneously, as a proof of concept. Dr. Ripoll enthuses, ‘Being able to analyse this data and actually see in 3D how a complex organism develops is very exciting, transporting traditional microscopy to a whole new level.’ Crucially, the speed can be further reduced for smaller volumes, allowing the imaging of 10 full volumes in one second. The practical implication of this is that fast processes such as the beating heart of the zebrafish, which were not possible to image under normal conditions, can now be captured in 3D and in slow motion. From observation to manipulation, for health benefits Enabling 3D imaging of large samples such as whole organs, quickly and efficiently, promises to impact on health related research. According to Dr. Ripoll, ‘These new approaches will provide a means to study gene expression and molecular function at the cellular level while looking at the organ as a whole, instead of imaging sections, offering complementary information not previously possible.’ The technology also offers the ability to follow in 3D, the fate of single cells, offering new ways to understand basic molecular biology which could potentially have direct implications for human health. In order to make this technology accessible, Dr. Ripoll has co-founded www.4dnature.eu (4D-Nature Imaging Consulting), a spin-off based on the patents generated during this project. For the team the next step is to go beyond passive observation of a 3-dimensional volume and towards actually influencing what is happening at the cellular level. This will have implications beyond in-vivo studies on model organisms to more general fast processes that occur in 3D, which will become amenable to control with sub micron voxel resolution.
Keywords
HIGH THROUGHPUT TOMO, Optical tomography, 3D imaging, microscopy, light beam scanning, tunable lenses, in-vivo, cellular control, gene expression
