Enhancing spatial and temporal resolution for isotropic volumetric imaging and 3D cell tracking

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Optical microscopy is ubiquitous in biological sciences with fluorescence microscopy in particular being utilised to map specific labelled proteins and/or structures. While the majority of such research is performed on populations of cells growing on glass slides, increasingly there is an appreciation that more realistic environments are required to obtain relevant data on biological processes, and ultimately this means using live biological models. A range of small, optically accessible, organisms (e.g. zebrafish, nematode worms, etc) provide convenient live samples for studying biological processes in vivo. Such samples are inherently three-dimensional (3-D), zebrafish being <1 mm in diameter when under 16 days old, and therefore require 3-D discrimination to provide unambiguous positional/structural information. Most 3-D microscopy is undertaken with laser scanning microscopes that scan a spot of light through the sample, building up a map of fluorescence intensity point by point. Such scanning microscopes are typically optimised for higher magnifications (i.e. small fields of view), suffer from unequal resolution (transverse better than axial) and require significant financial investment (e.g. often >£150K). An alternative method of acquiring 3-D data is optical projection tomography (OPT), the optical equivalent to X-ray computed tomography, which can be implemented on a standard wide-field imaging microscope and can provide 3-D imaging at a fraction of the cost of point scanning systems. In OPT, wide-field images (either fluorescence or transmitted light) of a rotating sample are acquired at different orientations. These images can be used to reconstruct the 3-D distribution of fluorescence/absorption. The standard approach to OPT imposes three key constraints: the requirement that at least the front half of the sample must be 'in focus', that the whole sample must stay in the field of view throughout the acquisition to prevent artefacts in the reconstruction process and that the sample must be non-scattering (i.e. transparent). The first two constraints limit the achievable spatial resolution, since they require the numerical aperture (NA) of imaging system to be small. This limit can be overcome by scanning the imaging lens, and therefore the focal plane, through the rotating sample while acquiring the angularly-resolved images. This produces an 'in focus' image of the whole sample that is superimposed on an out of focus "back-ground" signal that can be removed during image processing. We propose to extend this approach to yet higher resolution imaging of selected sub-volumes within the sample by incorporating a lateral scanning microscope stage to allow the motion of a "volume of interest" (VOI) inside a larger specimen (e.g. an organ) to be maintained in focus as the sample rotates. This VOI can then be modelled as a detailed structure within a larger 'unstructured' volume, to permit high resolution reconstruction without artefacts associated with parts of the sample entering/leaving the field of view. This would permit isotropic high resolution 3-D imaging of, e.g. immune cell distribution in specific organs in live zebrafish, which is currently not possible using the standard commercially available instruments.To address the limits to temporal resolution, we would investigate a novel "orthogonal scanning approach", acquiring sequential images at right-angles with respect to each other, such that the 3-D structure/location of features within the sample could be determined much faster than the rotation period. This could be applied, e.g. to follow cell migration within a live zebrafish. Finally we will extend this system to simultaneously acquire two images at different wavelengths of light using a commercially available spectral image splitter. By analysing these two wavelength channels we will be able to indirectly probe the signalling events that control the immune response and occur within cells.