3D cryo-imaging: a very high-resolution view of the whole mouse

Abstract

We developed the Case Cryo-imaging system that provides information rich, very high-resolution, color brightfield, and molecular fluorescence images of a whole mouse using a section-and-image block-face imaging technology. The system consists of a mouse-sized, motorized cryo-microtome with special features for imaging, a modified, brightfield/fluorescence microscope, and a robotic xyz imaging system positioner, all of which is fully automated by a control system. Using the robotic system, we acquired microscopic tiled images at a pixel size of 15.6 microm over the block face of a whole mouse sectioned at 40 microm, with a total data volume of 55 GB. Viewing 2D images at multiple resolutions, we identified small structures such as cardiac vessels, muscle layers, villi of the small intestine, the optic nerve, and layers of the eye. Cryo-imaging was also suitable for imaging embryo mutants in 3D. A mouse, in which enhanced green fluorescent protein was expressed under gamma actin promoter in smooth muscle cells, gave clear 3D views of smooth muscle in the urogenital and gastrointestinal tracts. With cryo-imaging, we could obtain 3D vasculature down to 10 microm, over very large regions of mouse brain. Software is fully automated with fully programmable imaging/sectioning protocols, email notifications, and automatic volume visualization. With a unique combination of field-of-view, depth of field, contrast, and resolution, the Case Cryo-imaging system fills the gap between whole animal in vivo imaging and histology.

Fig. 1

The Cryo-imaging system consists of a large section cryostat (s), microscope (m), low-noise camera (r), robotic positioner (p), illuminators (i), and control computer (c). During imaging sessions, the imaging system is automatically lowered inside the cryo chamber. An operator control box (arrow) allows manual interaction. Inset shows a specimen on the cryostat stage.

Fig. 2

Cryo-image of a whole adult mouse showing coronal section 448 out of 663 sections. The image was composited from 20 tiled acquisitions and has an original size of 5,300 × 2,100 pixels at 15.6 µm in-plane pixel size. The image background has been changed to black using automated image processing. Major organs like eyes, heart, lungs, liver, stomach, small intestine, and colon are easily identified. Note this compressed image shows clearly the right and left optic nerves, the rectus muscles of the eyes, the septa in the nose and the ribs. (Bar = 2 mm).

Fig. 3

High-resolution views from Fig. 2. (a) Section through heart, lungs, and livers (section 515). The left ventricle (V), atrium (A), bra chiocephalic trunk (B), right common carotid artery (C), right subcla vian artery (S), thoracic muscle (M), and iliocostal muscles (I) can be clearly distinguished. The ribs and hepatic vessels are visible as well. (b) Section through stomach (S) and small intestines (section 462). The characteristic layers of the stomach can be seen—lamina propria (LP), submucosa (arrow), and the muscularis externa (*). With a food-filled stomach the rugae are flattened. The villi (V) of the small intestine can be seen in different forms as the section goes through different cross-sectional planes. The food filling up the lumen gives it the comb-like appearance. Visceral fat (F) with fine blood vessels pack in between organs. (c) Left eye from section 448 showing optic nerve (O), sclera (S), choroids (C), retina (R), vitreous (V), and the lens (L). (Bar = 1 mm).

Fig. 4

3D visualization of cryo-images. (a) Volume visualization of the whole mouse from 2D sections shown in Fig. 2. A total of 13,260 individual images were used to create this true-color visualization. A cutaway shows views in three orthogonal planes. The coronal section was the cutting plane whereas the axial and sagittal sections have been digitally extracted. (b) 3D reconstruction from the manually segmented lungs with vasculature segmented through semi-automatic seeded region growing. (c) Segmentation-free volume visualization in which the same lungs (deep red) have been segmented automatically by optimizing the opacity for this tissue type. Skin was digitally removed to reveal organs like the brain, spinal cord and gastro-intestinal system. (inset) A color feature detector with combinational color and step opacity transfer function was used to automatically segment the stomach and intestines. (d) Low-resolution volume visualization of the liver with 3D surface reconstruction of hepatic vessels with a 3D zoom view (inset) showing a vessel branch at higher resolution.

Fig. 5

Cryo-imaging of microvessels in a mouse brain perfused with India ink. Low (left) and high (middle) magnification views show dark blood vessels. Vessels were segmented using an interactive 3D region growing algorithm. A 3D surface rendering of blood vessels (right) is shown. Vessels were segmented down to a size of about 10 µm in diameter. (Bar = 250 µm).

Fig. 6

Fluorescence imaging. 3D reconstruction from a fluorescent image stack of an adult transgenic mouse where the smooth muscles of the gastrointestinal and genitourinary system express EGFP. Note the continuity of the GI system ending in the rectum and the external anal sphincter muscle (R). The convolutions of the pair of epididymis (E) can also be seen. The 2D brightfield image is representative of the anatomical context that the brightfield image stack provides to the fluorescent volume.

Fig. 7

Embryo imaging. The cryo-imaging system is also capable of imaging smaller specimens such as mouse embryos. (a) shows a brightfield cryo-image of a stage E.16, wildtype (left) and a Cited2^−/− mutant (right) mouse embryo for phenotypical comparison. Anatomical details such as the spinal cord, vertebrae, ribs, parts of the brain (medulla oblongata, pons, midbrain, forebrain), liver, and the intestines can be clearly seen in both the images. The wildtype has an adrenal gland (see arrow and cropped inset magnified 300%) located above the kidney (K) whereas the mutant does not which is often associated with this genotype (Bar = 3 mm). (b) Automatic volume visualization of the Cited2 ^−/− mutant mouse embryo using color channel specific opacity transfer functions to visualize dominant red in heart, liver, and vasculature and white in spine. The overlaid 2D cryo-image, showing anatomy, was automatically segmented from the embedding medium. (c) 3D reconstruction of GFP-labeled smooth muscles in an E16.5 SMGA/EGFP mouse embryo for the transgenic adult shown in Fig. 6. The airway smooth muscles of the lungs and the smooth muscles of the GI and GU system are clearly seen in this surface rendering showing the morphology and continuity of the organ system. The bright 2D cryo-image may be used for anatomical correlation.