Three-dimensional mouse brain cytoarchitecture revealed by laboratory-based x-ray phase-contrast tomography.

Sample preparation

The brain preparation is based on dehydration of paraformaldehyde-fixed material in ethanol, removal of lipids in xylene and evaporation of xylene. We noted that the contrast was higher when dehydration was performed via transcardial perfusion with ethanol instead of conventional dehydration by immersion in ethanol. All experiments were in accordance with German ethical guidelines for the care and use of laboratory animals in experiments and approved by the local authorities (Landesverwaltungsamt Sachsen-Anhalt). Twenty adult mice were used for developing the paraformaldehyde/ethanol perfusion protocol. For brain preparation, adult mice were deeply anesthetized (induced with 2% Isoflurane, 0.75% O 2 , 0.55% N 2 O, followed by i.p. injection of 150 μ l of a Ketamin/Xylazin solution containing 12.5 mg Ketamin/ml and 0.5% Xylazin (Ketamin: Ratiopharm, Germany, Xylazin/Rompun: Bayer Healthcare, Germany)) and transcardially perfused with a short flush of saline (5 ml) followed by 15 min of phosphate buffered (100 mM, pH 7.4) 4% paraformaldehyde solution and 30 min with ethanol (≥99.8%, containing 1% methylethylketone, Carl Roth, Germany, termed ‘100% ethanol’ in the following according to common practice in histology). A peristaltic pump was used for perfusion. Flow rates were 15 ml/min during the first three minutes of perfusion (including perfusion with saline) and reduced to 7 ml/min during the remaining period. Animals were perfused with the different solutions without interrupting the flow. Brains were removed after perfusion and stored in 100% ethanol overnight in a refrigerator (7 °C). Brains were then transferred to a fresh solution of 100% ethanol and remained stored in this solution in the refrigerator for one to three days. Entire brains, or parts of the brains were then transferred to xylene (≥99%, p.a., isomers, for use in histology, Carl Roth, Germany). After overnight storage in xylene at room temperature the material was transferred to a fresh xylene solution and stored in this solution for one to two days at room temperature. The material was then placed on tissue paper under a hood at room temperature and dried overnight. For the experiments, the samples were carefully squeezed into a pipette tip to keep the sample position stable during the scan. In order to prevent sample changes due to external influences, a layer of hematocrit was deposited above the opening of the pipette tip, creating a closed chamber and therefore maintaining experimental conditions ( Fig. 1c ). During development of the protocol brains not used for CT experiments were sometimes stored for several weeks in ethanol. We did not note any changes in drying behavior or gross morphology. Some of these brains were rehydrated ( Supplementary Fig. S6 ). Rehydration times may depend on storage times in the different solutions and in the dried state. This was not systematically tested. No changes in macroscopical appearance nor in contrast were observed when entire brains were dried as compared to smaller parts of the brain. Preparation of small-sized specimen suitable for mounting on object holders for CT was easier when samples were prepared before drying. We also noted that the entire procedure is applicable to large specimen (e.g. whole mouse bodies as the largest tested specimen). Evaporation of xylene resulted in tissue shrinkage but did not alter the gross morphology ( Supplementary Fig. S6 ).

Laboratory setup

A sketch of the generic laboratory setup is shown in Fig. 1 . The liquid-metal jet source (Excillum, Stockholm, Sweden) consists of Galinstan (68.5% Ga, 21.5% In und 10% Sn) with the characteristic photon energy 9.25 keV (Ga- K α ). For the experiments, it was operated at a tube voltage of 40 kV and the electrons were focussed to a size of 10 × 40 μ m 2 , resulting in an approximate focal spot size of 10 × 10 μ m 2 . The sample is positioned on a sample tower with three translational and one rotational axes for tomographic measurements. Additionally, two translations parallel and perpendicular to the beam allow for positioning of the rotation axis with respect to the optical axis. For alignment of the pitch angle and the zero position of the rotational axis, the detector is placed on a stage with two translations perpendicular to the optical axis. In the inverse geometry the lens-coupled scintillator-based camera XSight (Rigaku, Tokyo, Japan) was introduced in the setup. It consists of a thin single-crystal scintillator, a 10-fold magnification objective and a 2504 × 3326 pixels CCD chip with a resulting pixel size of 0.54 μ m. For the experiments in cone-beam geometry a Flat Panel CMOS detector with a GdOS:Tb-scintillator screen (PerkinElmer, Waltham, USA) was installed. It consists of 1536 × 1944 pixels with a pixel size of 74.8 μ m. The geometric as well as experimental parameters for the measurements are listed in Supplementary Tab. S2 . Data processing (laboratory setup) Phase retrieval was performed with the BAC algorithm for all recorded projections using the parameters listed in Supplementary Table S2 . Prior to the reconstruction of the 3D volume a wavelet-based ring removal algorithm was applied to the sinograms 29 . The 3D reconstruction of the volume was obtained with the UltraFast ConeBeam Reconstruction Software (Bronnikov Algorithms, Arnhem, The Netherlands). The visualization of the data was carried out with Avizo (FEI Visualization Sciences Group, Burlington, USA). The pink colorcode was chosen so that the virtual slices resemble histological sections stained with hematoxylin and eosin (H&E stain). The segmentation of the single purkinje cells was performed manually for a small part of the data set, whereas for the labeling of the cells within the molecular and granular layer a semi-automatical approach was used. Here, the cells were labeled with a threshold-based brush tool that, within the manually selected region by the user, only marks pixels with a gray value within a given range. Setup ID19 (ESRF) The experiments were carried out at the ID19 beamline (ESRF, Grenoble, France) with a parallel beam geometry. The sample is positioned approximately 145 m behind the undulator on a sample tower with three translational and one rotational axes, while the position of the rotational axis can be adjusted via two additional translations. The energy of the setup is variable and for this experiment chosen as 18.685 keV with a flux of . The intensity distribution behind the sample is recorded with a lens-coupled scintillator based FReLoN ( F ast Re adout Lo w N oise) CCD camera (ESRF, Grenoble, France) which is equipped with an optic revolver system (Optique Peter, Lentilly, France). The 10 μ m GGG:Eu scintillator converts the x-rays to optical light which reaches the CCD placed perpendicular to the optical axis via a mirror. It consists of 2048 × 2048 pixels with a size of 14 μ m which can be modified by the choice of objective directly behind the scintillator as well as eye-piece directly before the CCD, leading to a minimum pixel size of 280 nm. For the experiments shown here, we chose a pixel size of 0.7 μ m. To image the whole sample 12 individual tomograms at different positions on the sample were recorded and virtually stitched together. For stitching, the individual reconstructed subvolumes were loaded into the the software Avizo and overlapping parts between adjacent volumes were determined by looking for specific landmarks appearing in both datasets. After finding those landmarks the volumes were visually aligned and merged via the standard merging function in Avizo. The experimental parameters for a single tomogram are listed in Supplementary Table S2 . Data processing (ID19) As for the laboratory setup, phase retrieval was performed with the BAC algorithm for all recorded projections using the parameters listed in Supplementary Table S2 . Prior to the 3D reconstruction a simple ring removal algorithm was applied 30 which already delivered satisfying results and is significantly faster than the one used for the laboratory data. For the tomographic reconstruction the Matlab (Mathworks, Natick, USA) implemented function iradon , which performs a simple filtered backprojection, was used with the standard Ram-Lak filter. The analysis of the 3D data was again carried out with Avizo and the colorcode was chosen similar to the laboratory results. For the segmentation of the granular layer, the structure was labeled manually in approximately every third layer, followed by an interpolation by the software. Afterwards the labels were smoothed with a custom Matlab script and multiplied with the 3D data set, leaving only non-zero gray values within the granular layer. To get a 3D visualization of this data, a volume rendering was performed with a colormap, where regions outside of the granular layer are displayed as transparent. The cellular segmentation was performed in the same way as for the laboratory setup, again only for a small part of the sample ( Supplementary Video 2 ).

Supplementary Material Supplementary Information Supplementary Video 1 Supplementary Video 2