A bold new effort to disrupt every gene in the mouse genome necessitates systematic, interdisciplinary approaches to analyzing patterning defects in the mouse embryo. We present a novel, rapid, and inexpensive method for obtaining high-resolution virtual histology for phenotypic assessment of mouse embryos. Using osmium tetroxide to differentially stain tissues followed by volumetric X-ray computed tomography to image whole embryos, isometric resolutions of 27 μm or 8 μm were achieved with scan times of 2 h or 12 h, respectively, using mid-gestation E9.5–E12.5 embryos. The datasets generated by this method are immediately amenable to state-of-the-art computational methods of organ patterning analysis. This technique to assess embryo anatomy represents a significant improvement in resolution, time, and expense for the quantitative, three-dimensional analysis of developmental patterning defects attributed to genetically engineered mutations and chemically induced embryotoxicity.

We have the studied the binding of 5- ((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 µm diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. We have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. We estimate the site number to be ≈1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is ≈(1.3 ( 0.3) × 10 7 M -1 s -1 . The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s -1 shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another result in self‐quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity of antibodies relative to one another on a highly labeled cell surface. Since self‐quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase‐sensitive detection method to investigate the impact of self‐quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti‐mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self‐quenching effects expected at high densities of FITC molecules. (This article is a U.S. Government work and, as such, is in the public domain n the United States of America.) © 1996 Wiley‐Liss, Inc.

Contrast-enhanced small-animal computed tomography is an economical and highly quantitative tool for serially examining tumor development in situ, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s). We present practical considerations for visualizing the vascular network of transgenic mouse tumors. Using a long-acting iodinated triglyceride blood-pool contrast agent, we present optimized scanner acquisition parameters and volume-rendering techniques for examining the intermediate and large vessels of complex spontaneous tumors (e.g., alveolar rhabdomyosarcomas) in transgenic mice. Our findings indicate that multiple-frame, 360-720 view acquisitions were mandatory for clarifying bone and soft tissue from vessel contrast. This finding was consistent in visualizations using a one-dimensional transfer function where voxel color and opacity was assigned in proportion to CT value and a two-dimensional transfer function where voxel color and opacity was assigned in proportion to CT value and gradient magnitude. This study lays a groundwork for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic mice.

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