Grant J. Steyer

3D Cryo-Imaging: A Very High-Resolution View of the Whole Mouse

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 μm over the block face of a whole mouse sectioned at 40 μm, 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 μm, 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. histology. Anat Rec, 292:342–351, 2009.

Whole mouse cryo-imaging

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.

Visualization of color anatomy and molecular fluorescence in whole-mouse cryo-imaging

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 low-resolution 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.

Detection and quantification of fluorescent cell clusters in cryo-imaging

We developed and evaluated an algorithm for enumerating fluorescently labeled cells (e.g., stem and cancer cells) in mouse-sized, microscopic-resolution, cryo-image volumes. Fluorescent cell clusters were detected, segmented, and then fit with a model which incorporated a priori information about cell size, shape, and intensity. The robust algorithm performed well in phantom and tissue imaging tests, including accurate (<2% error) counting of cells in mouse. Preliminary experiments demonstrate that cryo-imaging and software can uniquely analyze delivery, homing to an organ and tissue distribution of stem cell therapeutics.

Removal of subsurface fluorescence in cryo-imaging using deconvolution

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.

Multi-scale characterization of the PEPCK-C mus mouse through 3D Cryo-imaging

We have developed, for the Case 3D Cryo-imaging system, a specialized, multiscale visualization scheme which provides color-rich volume rendering and multiplanar reformatting enabling one to visualize an entire mouse and zoom in to organ, tissue, and microscopic scales. With this system, we have anatomically characterized, in 3D, from whole animal to tissue level, a transgenic mouse and compared it with its control. The transgenic mouse overexpresses the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C) in its skeletal muscle and is capable of greatly enhanced physical endurance and has a longer life-span and reproductive life as compared to control animals. We semiautomatically analyzed selected organs such as kidney, heart, adrenal gland, spleen, and ovaries and found comparatively enlarged heart, much less visceral, subcutaneous, and pericardial adipose tissue, and higher tibia-to-femur ratio in the transgenic animal. Microscopically, individual skeletal muscle fibers, fine mesenteric blood vessels, and intestinal villi, among others, were clearly seen.

Calcium imaging of sonoporation of mammalian cells

Ultrasound mediated delivery of compounds is a relatively recent development in drug delivery and gene transfection techniques. Due to the lack of methods for real‐time monitoring of sonoporation at the cellular level, the efficiency of drug/gene delivery and sonoporation associated side effects, such as the loss of cell viability and enhanced apoptosis, have been studied only through post US exposure analyses, requiring days for cell incubation. Furthermore, because microporation appears to be transient in nature, it was not possible to correlate transfection with microporation on an individual cellular basis. By studying the role of calcium in the cell and using fluorescent calcium imaging to study sonoporation it is possible to quantify both cell porosity and sonoporation side effects. Since both post sonoporation cell survival and delivery efficiency are related to the dynamic process of the cell membrane poration, calcium imaging of sonoporation will provide important knowledge to obtain improved und...

Sub-Micron 3D Fluorescent Imaging and Visualization of Remodeling Cavities in Cancellous Bone

The mechanical properties of cancellous bone are determined from a combination of bone quantity (volume), the material properties of the mineralized tissue, and microarchitecture. Bone remodeling is the primary process through which bone mass and structure are altered in the adult skeleton. Bone remodeling involves the coordinated activity of osteoclast and osteoblast cells, which resorb and then form bone at an isolated location on the cancellous bone surface. Because bone resorption precedes formation, each bone remodeling event in cancellous bone is associated with a temporary void on the bone surface known as a remodeling cavity. It has been proposed that remodeling cavities can act as stress risers, modifying stress distributions in cancellous bone and potentially impairing bone strength, stiffness and other mechanical properties. While high resolution finite element modeling supports the idea that remodeling cavities have the potential to modify mechanical properties at the micro-scale (in individual trabeculae) [1] and at the apparent level (entire cancellous bone specimens)[2, 3], the experiments required to confirm these findings are limited because a repeatable method of quantifying the number and size (length width and depth) of remodeling cavities in entire cancellous bone specimens has not yet been demonstrated.Copyright