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 mm over the block face of a whole mouse sectioned at 40 mm, 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 mm, 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.

We developed a cryo-imaging system, which alternates between sectioning (10–40 μm) and imaging bright field and fluorescence block-face image volumes with micronscale-resolution. For applications requiring single-cell detection of fluorescently labeled cells anywhere in a mouse, we are developing software for reduction of out-of-plane fluorescence. In mouse experiments, we imaged GFP-labeled cancer and stem cells, and cell-sized fluorescent microspheres. To remove out-of-plane fluorescence, we used a simplified model of light-tissue interaction whereby the next-image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing an objective function on subtracted images. Tissue-specific attenuation parameters [μT: heart (267 ± 47.6 cm−1), liver (218 ± 27.1 cm−1), brain (161 ± 27.4 cm−1)] were found to be within the range of estimates in the literature. “Next-image” processing removed out-of-plane fluorescence equally well across multiple tissues (brain, kidney, liver, etc.), and analysis of 200 microsphere images gave 97 ± 2% reduction of out-of-plane fluorescence. Next-image processing greatly improved axial-resolution, enabled high quality 3D volume renderings, and improved automated enumeration of single cells by up to 24%. The method has been used to identify metastatic cancer sites, determine homing of stem cells to injury sites, and show microsphere distribution correlated with blood flow patterns.

We compared image restoration methods [Richardson-Lucy (RL), Wiener, and Next-image] with measured “scatter” point-spread-functions, for removing subsurface fluorescence from section-and-image cryo-image volumes. All methods removed haze, delineated single cells from clusters, and improved visualization, but RL best represented structures. Contrast-to-noise and contrast-to-background improvement from RL and Wiener were comparable and 35% better than Next-image. Concerning detection of labeled cells, ROC analyses showed RL ≈Wiener > Next-image >> no processing. Next-image was faster than other methods and less prone to image processing artifacts. RL is recommended for the best restoration of the shape and size of fluorescent structures.

The Case cryo-imaging system is a section and image system which allows one to acquire micron-scale, information rich, whole mouse color bright field and molecular fluorescence images of an entire mouse. Cryo-imaging is used in a variety of applications, including mouse and embryo anatomical phenotyping, drug delivery, imaging agents, metastastic cancer, stem cells, and very high resolution vascular imaging, among many. Cryo-imaging fills the gap between whole animal in vivo imaging and histology, allowing one to image a mouse along the continuum from the mouse → organ → tissue structure → cell → sub-cellular domains. In this overview, we describe the technology and a variety of exciting applications. Enhancements to the system now enable tiled acquisition of high resolution images to cover an entire mouse. High resolution fluorescence imaging, aided by a novel subtraction processing algorithm to remove sub-surface fluorescence, makes it possible to detect fluorescently-labeled single cells. Multi-modality experiments in Magnetic Resonance Imaging and Cryo-imaging of a whole mouse demonstrate superior resolution of cryo-images and efficiency of registration techniques. The 3D results demonstrate the novel true-color volume visualization tools we have developed and the inherent advantage of cryo-imaging in providing unlimited depth of field and spatial resolution. The recent results continue to demonstrate the value cryo-imaging provides in the field of small animal imaging research.

We developed multi-scale, live-time interactive visualization of color image data, including microscopic whole-mouse cryo-images serving many biomedical applications. Using true-color volume rendering, we interactively, selectively enhanced anatomy using feature detection. For example, to enhance red organs (vessels, liver, etc.) and internal surfaces, we computed a red feature from R/(R+G+B) and surface features from color/gray-scale gradients, respectively. For >70GB cryo-image volumes, we developed multi-resolution visualization, which provided lowresolution rendering of an entire mouse and zooming to organs, tissues, and cells. Fusions of fluorescence and color cryo-volumes uniquely showed biodistribution of metastatic and stem cells within an anatomical context.