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1.
Int J Biomed Imaging ; 2012: 698413, 2012.
Article in English | MEDLINE | ID: mdl-22481905

ABSTRACT

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.

2.
Comput Med Imaging Graph ; 35(3): 195-205, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21041065

ABSTRACT

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.


Subject(s)
Colorimetry/methods , Colorimetry/veterinary , Cryopreservation/veterinary , Image Interpretation, Computer-Assisted/methods , Microscopy, Fluorescence/methods , Microscopy, Fluorescence/veterinary , Whole Body Imaging/methods , Whole Body Imaging/veterinary , Animals , Image Enhancement/methods , Mice , Reproducibility of Results , Sensitivity and Specificity
3.
Opt Express ; 18(21): 22324-38, 2010 Oct 11.
Article in English | MEDLINE | ID: mdl-20941133

ABSTRACT

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.


Subject(s)
Biotechnology/methods , Diagnostic Imaging/methods , Freezing , Microscopy, Fluorescence/methods , Optics and Photonics , Algorithms , Animals , Cryopreservation , Green Fluorescent Proteins/metabolism , Humans , Image Processing, Computer-Assisted , Mice , Microspheres , Models, Statistical , Phantoms, Imaging
4.
Int J Biomed Imaging ; 2010: 105984, 2010.
Article in English | MEDLINE | ID: mdl-20467563

ABSTRACT

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.

5.
Proc SPIE Int Soc Opt Eng ; 7262: 72620W-72620W8, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19756213

ABSTRACT

We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryo-imaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 µm) and imaging, collecting color brightfield and fluorescent block-face image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface 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 entropy of subtracted images. Tissue specific attenuation parameters were found [u(T) : heart (267 ± 47.6 µm), liver (218 ± 27.1 µm), brain (161 ± 27.4 µm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97±2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns.We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron-scale, fluorescence, and bright field image data. Here we describe our image pre-processing, analysis, and visualization techniques. Processing improves axial resolution, reduces subsurface fluorescence by 97%, and enables single cell detection and counting. High quality 3D volume renderings enable us to evaluate cell distribution patterns. Applications include the myriad of biomedical experiments using fluorescent reporter gene and exogenous fluorophore labeling of cells in applications such as stem cell regenerative medicine, cancer, tissue engineering, etc.

6.
Proc SPIE Int Soc Opt Eng ; 7262: 72655V, 2009.
Article in English | MEDLINE | ID: mdl-19606273

ABSTRACT

We are developing enhanced volume rendering techniques for color image data. One target application is cryo-imaging, which provides whole-mouse, micron-scale, anatomical color, and molecular fluorescence image volumes by alternatively sectioning and imaging the frozen tissue block face. With the rich color images provided by cryo-imaging, we use true-color volume rendering and visually enhance anatomical regions by proper selection of voxel opacity. To compute opacity, we use color and/or gradient feature detection followed by suitable opacity transfer functions (OTF). An interactive user interface allows one to select from among multiple color and gradient feature detectors, OTF's, and their associated parameters, and to compute in live time new volume visualizations from within the Amira platform. We are also developing multi-resolution volume rendering techniques to accommodate extremely large (>60GB) cryo-image data sets. Together, these enhancements enable us to interactively interrogate cryo-image volume data and create useful renderings with "implicit segmentation" of organs.

7.
Ann Biomed Eng ; 37(8): 1613-28, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19513848

ABSTRACT

We developed a cryo-imaging system, which alternates between sectioning (10-40 microm) and imaging bright field and fluorescence block-face image volumes with micron-scale-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 [micro(T): 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.


Subject(s)
Carcinoma, Lewis Lung/pathology , Freezing , Image Processing, Computer-Assisted/instrumentation , Image Processing, Computer-Assisted/methods , Microscopy, Fluorescence, Multiphoton/instrumentation , Microscopy, Fluorescence, Multiphoton/methods , Microspheres , Stem Cells/pathology , Tibial Fractures/pathology , Animals , Cell Line, Tumor , Mice , Mice, Transgenic , Neoplasm Metastasis
8.
Anat Rec (Hoboken) ; 292(3): 342-51, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19248166

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.


Subject(s)
Diagnostic Imaging , Image Processing, Computer-Assisted , Imaging, Three-Dimensional/methods , Whole Body Imaging , Actins/genetics , Animals , Automation , Fluorescence , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Smooth , Promoter Regions, Genetic
9.
Proc SPIE Int Soc Opt Eng ; 6916: 69161I-69161I9, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-19756215

ABSTRACT

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.

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