Abstract
We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.
Highlights
Understanding biological systems necessitates studying the spatial distribution of cells and their temporal dynamics
A series of tracks for individual neutrophils in the fish tail were determined from the projection images, as described above, with the Z axis approximately aligned along the length of the fish
In conclusion, we have demonstrated a technique for mesoscopic 3-D cell tracking combined with optical projection tomography (OPT) that utilizes multiplexed angular projections to realize a time lapse resolution determined by the delay between sequential projection images rather than the total volumetric acquisition time
Summary
Understanding biological systems necessitates studying the spatial distribution of cells and their temporal dynamics. The optical transparency of zebrafish allows visualization of physiological and pathological processes in the intact organism, which can provide insights into the global response of organisms to insults and disease. This has motivated two-dimensional [1,2,3] and three dimensional [4,5,6,7] imaging studies of in vivo cell migration in sub regions within zebrafish using fluorescence microscopy but to date there has been no report of studies of 3-D time-lapse cell-tracking techniques throughout the whole volume of a fish
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