In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography

Three-dimensional optical imaging is increasingly being used in many areas of biology, allowing the analysis of complex and diverse models. Aside from commonly used confocal and multiphoton microscopies, other three-dimensional imaging techniques, such as single-plane illumination microscopy¹ and optical projection tomography (OPT)² are emerging as valuable tools for a wide range of biological applications that require the visualization of intact and entire organisms or biological tissues. OPT is a relatively simple and low-cost technique that is particularly suitable for studying millimeter-sized samples. Similarly to x-ray computed tomography, OPT is based on the acquisition of a sequence of optical transmission (or fluorescence) images of the sample at several orientations. The acquired images, or projections, are combined to reconstruct the 3-D volume of the sample, typically using a backprojection algorithm. OPT is currently evolving from a method to image small chemically cleared samples to a three-dimensional imaging technique for living specimens.^3,4,5

In this paper, we report a new contrast mechanism in OPT, given by the movement of cells present in bloodstream. Looking at a living transparent or weakly scattering sample it is possible to observe the flow of the blood cells. Therefore, by acquiring several time frames of the specimen and applying a motion-analysis algorithm, it is possible to obtain a map of the sample vasculature.⁶ Here, we show that by mathematical processing the vascular maps obtained at different angles it is possible to produce and visualize a 3-D casting of the vasculature of the specimen, noninvasively and without the need for any fluorescent probe. This results in a low-cost, label-free, three-dimensional imaging technique, which we will hereafter call flow-OPT. The present study has been performed on a juvenile zebrafish (Danio rerio), a model organism widely used in developmental biology⁷ and oncology.⁸ However, these concepts could be generally extended to other transparent and weakly scattering living samples.

The flow-OPT system is depicted in Fig. 1. The light emitted by a high-power white light-emitting diode (MCWHL2, Thorlabs, Delaware) is passed through a diffuser (DG10-1500-MD, Thorlabs, Delaware) and projected on the specimen with a telecentric lens (TC2309, OptoEngineering, IT). The light transmitted through the sample is imaged by a 5X telecentric objective (NT56-986, Edmund Optics, Delaware) on a CMOS camera (B771U, Pixelink, California), which is operated at half resolution (640 × 480 pixels) to acquire 100 fps. Telecentric optics are used in order to keep a constant magnification through the specimen.