Quantitative Biology Core, consisting of a phosphorimager, a CCD camera/image analysis system, and a spectrofluorometer with stopped-flow capability will be established for the use of biological scientists in the College of Arts and Science at Vanderbilt University. The main objective in the establishment of this resource is to enable the participating scientists, and the undergraduate, graduate, and postdoctoral students in their laboratories, to obtain quantitative data, currently unobtainable due to the lack of access to appropriate equipment. The phosphorimager will allow quantitation of data presently collected by autoradiography. Autoradiography is an inherently non-quantitative technique. Exposure of a grain of film emulsion requires two photons. Such two-photon processes under-report low levels of activity, because at low levels, a high proportion of emulsion grains will have interacted with only one photon and therefore remain unexposed on development; and they under-report high levels of activity, because a high proportion of photons interact with grains that have already been fully exposed. Sensitivity to the low end can be enhanced by pre flashing the film, but whether pre-flashed or not, the linear response range is narrow, perhaps one log. In contrast, the proposed phosphorimager can collect quantitative data over a four log range, and collect it an order of magnitude faster than exposure of film. This instrument will be applied to a wide range of studies, for example, the quantitation of radiolabeled DNA, RNA, and proteins separated on gels. The CCD camera/image analysis system will allow quantitation of data presently collected qualitatively by photomicroscopy. For example, immunofluorescence localization studies can be carried out on this system with a resolution approaching that of confocal microscopy at a fraction of the cost. A software-controlled stepper motor can be used to acquire images from sequential optical planes, effectively optically sectioning the specimen. Digitization of each field allows quantitative analysis of the fluorescent marker in that field, which, combined with the optical sectioning, results in quantitation within a predetermined volume within the specimen. This facility will be employed in immunolocalization studies in yeast, transgenic Drosophilia at various developmental stages, Ascidian larvae, Dictyostelium cells, and mammalian neuronal cells and tissues, and in studies of the assembly of microtubules in vitro. The spectrofluorometer will allow collection of quantitative fluorescence intensity and kinetic data not currently accessible with equipment available in the College of Arts and Science at Vanderbilt. For example, utilization of the stopped-flow accessory with the T- format spectrofluorometer configured with excitation and emission polarizers, for simultaneous acquisition of parallel and perpendicular emission with polarized excitation, will allow real-time acquisition of the association kinetics of nascent RNA transcripts with proteins that make up hnRNP particles, peptide ligands with their cell surface receptors, and regulatory proteins with Ca++-release channels, and of tubulin into microtubules.
|Effective start/end date||1/15/95 → 12/31/96|
- National Science Foundation: $167,980.00