Quantitative Biology Core (phosphorimager/CCD camera-image analysis system/spectro-fluorometer)

将建立定量生物学核心，由磷光成像仪、CCD照相机/图像分析系统和具有停流能力的分光荧光计组成，供范德比尔特大学文理学院的生物科学家使用。 建立这一资源的主要目标是使参与的科学家以及实验室的本科生、研究生和博士后学生能够获得目前由于缺乏适当设备而无法获得的定量数据。 磷光成像仪将允许对目前通过放射自显影术收集的数据进行定量。放射自显影是一种固有的非定量技术。曝光一粒胶片乳剂需要两个光子。 这种双光子过程低估了低水平的活性，因为在低水平下，高比例的乳剂颗粒将仅与一个光子相互作用，因此在显影时保持未曝光;并且它们低估了高水平的活性，因为高比例的光子与已经完全曝光的颗粒相互作用。对低端的灵敏度可以通过预闪光胶片来增强，但是无论预闪光与否，线性响应范围都很窄，可能只有一个对数。相比之下，所提出的磷光成像仪可以收集超过四个对数范围的定量数据，并且收集它比胶片曝光快一个数量级。该仪器将被应用于广泛的研究，例如，定量的放射性标记的DNA，RNA和蛋白质上分离的凝胶。 CCD照相机/图像分析系统将允许对目前通过显微照相术定性收集的数据进行定量。例如，免疫荧光定位研究可以在该系统上进行，其分辨率接近共聚焦显微镜的分辨率，成本仅为一小部分。软件控制的步进电机可以用于从顺序的光学平面获取图像，有效地光学地 切片标本。每个场的数字化允许对该场中的荧光标记物进行定量分析，其与光学切片相结合，导致在样本内的预定体积内进行定量。该设施将用于酵母、处于不同发育阶段的转基因果蝇、海鞘幼虫、网骨藻细胞和哺乳动物神经元细胞和组织的免疫定位研究，以及体外微管组装的研究。 荧光分光光度计将允许收集定量荧光强度和动力学数据，目前无法使用范德比尔特文理学院的设备。 例如，利用具有配置有激发和发射偏振器的T格式荧光分光光度计的停流附件，用于同时采集具有偏振激发的平行和垂直发射，将允许实时采集新生RNA转录物与构成hnRNP颗粒的蛋白质、肽配体与其细胞表面受体的缔合动力学，和具有Ca++释放通道的调节蛋白，以及微管蛋白进入微管的调节蛋白。

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.