Abstract
BackgroundCurrent mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples—for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples—cannot be assessed. We have developed “ALMOST,” a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters.ResultsAs well as demonstrating image formation, we provide background information and explain the reconstruction—and consequent rendering—using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole.ConclusionThus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.
Highlights
Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays
As light sheet microscopy uses a perpendicular illumination scheme with respect to the light detection, we presumed it would not be the best option for reflective light detection
It contains a reflective background via white paper and aluminum foil, a non-coherent unfocused light source of LED goosenecks directed at the sample, and a diffuser made of milk glass placed between the illuminator and the sample (Fig. 1c, e)
Summary
Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples—for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples—cannot be assessed. The 3D modality of these methods differ, with light sheet microscopy directly generating a stack of confocal sections and OPT acquiring multiple images collected at different angles by rotating the sample with respect to. Both light sheet and OPT devices require transparent samples to image fluorescence within the tissue. Apart from inherently transparent biological samples, like the zebrafish (Danio rerio), transparency needs to be induced using chemical “clearing” methods
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