High resolution optical projection tomography platform for multispectral imaging of the mouse gut

Abstract

Optical projection tomography (OPT) is a powerful tool for three-dimensional imaging of mesoscopic biological samples with great use for biomedical phenotyping studies. We present a fluorescent OPT platform that enables direct visualization of biological specimens and processes at a centimeter scale with high spatial resolution, as well as fast data throughput and reconstruction. We demonstrate nearly isotropic sub-28 µm resolution over more than 60 mm³ after reconstruction of a single acquisition. Our setup is optimized for imaging the mouse gut at multiple wavelengths. Thanks to a new sample preparation protocol specifically developed for gut specimens, we can observe the spatial arrangement of the intestinal villi and the vasculature network of a 3-cm long healthy mouse gut. Besides the blood vessel network surrounding the gastrointestinal tract, we observe traces of vasculature at the villi ends close to the lumen. The combination of rapid acquisition and a large field of view with high spatial resolution in 3D mesoscopic imaging holds an invaluable potential for gastrointestinal pathology research.

Fig. 1.

OPT design. a) Optical layout superimposed with the corresponding mounting system (view from the top). Optical components are numbered from 1 to 11. The optical path of the excitation and detection beams are represented in purple and green, respectively. b) A three-quarter view of the motorized sample positioning stages, as well as the transparent cuvette filled with BABB. c) Photograph taken from the side of the cuvette. A 30 mm long mouse gut sample is embedded into a cylinder of agarose gel submerged in the BABB cuvette. The gel cylinder is glued to the sample holder, which is held together with the rotating motor using a magnet. The gut sample is excited by a LED centered at 415 nm and the fluorescent signal is acquired by the camera for multiple angular positions of the specimen. d) A selection of five projections showing the characteristic acquisitions of cleared samples over 360° in OPT imaging.

Fig. 2.

OPT PSF characterization a) Single projection of the acquired fluorescent microspheres. The close-up shows an in-focus microsphere (scale bar = 20 µm). b) Y-projection over the reconstructed volume of microspheres to show the XZ-plane PSF. The close-up shows a typical microsphere (scale bar = 20 µm). c) Gaussian fit of the PSF integral represented in a) along the Y-axis. The given value is the Gaussian FWHM. d) Gaussian fits of the PSF integrals represented in b) along the X- and Z-axis. The given value is the Gaussian largest FWHM. e) XZ-plane lateral FWHM of the reconstructed PSF as a function of the distance to the rotation axis for multiple angular resolutions. Each data point is the mean of microspheres included in a 0.1 mm interval. f) XZ-plane mean lateral resolution (black line) measured from the reconstructed PSF of fluorescent microspheres as a function of the distance of the rotation axis to the focal plane. 15 PSFs (blue dots) are represented by axis position. A typical PSF for each position is represented at the top of the graph. A seagull shape of the PSF is visible as the distance of the rotation axis to the focal plane increases (scale bar = 20 µm).

Fig. 3.

Mouse small intestine portion imaged by multispectral OPT. For a better 3D experience, see the movie sequence shown in Visualization 1. a) 3D blend rendering of a 3-cm long mouse gut portion acquired using 2 spectral channels. The vasculature (magenta) is stained using an anti-CD31 antibody combined with an anti-rat Alexa Fluor 647 secondary antibody and is excited at 625 nm. The tissue is imaged using autofluorescence of flavin adenine dinucleotide excited at 415 nm. b) Sliced perspective view of the gut portion emphasizing the intestinal villi structure, as well as the penetrating vessels, and highlighting the 3D advantage of the technique. c) OPT cross-section view of the gut showing the submucosa and muscularis layers of the tissue surrounding the villi structures. d) Close-up of the typical villi structure with the increased concentration of the vasculature visible close to the lumen as well as in the submucosa and muscularis layers.