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

将为范德比尔特大学文理学院的生物科学家建立定量生物学核心，由一个磷成像仪、一个CCD相机/图像分析系统和一个具有止流能力的荧光光谱仪组成。建立这一资源的主要目标是使参与的科学家以及他们实验室的本科生、研究生和博士后学生能够获得定量数据，这些数据目前由于缺乏适当的设备而无法获得。该磷光成像仪将允许对目前由放射自显影术收集的数据进行定量。放射自显影术本质上是一种非定量技术。一粒胶片乳剂的曝光需要两个光子。这种双光子过程低估了低水平的活性，因为在低水平下，很大比例的乳剂颗粒只与一个光子相互作用，因此在发育过程中保持不暴露；而且他们低估了高水平的活动，因为很大比例的光子与已经完全暴露的颗粒相互作用。对低端的灵敏度可以通过预闪膜来增强，但无论是否预闪，线性响应范围都很窄，可能只有一个对数。相比之下，所提出的磷成像仪可以在4个对数范围内收集定量数据，并且收集速度比胶片曝光快一个数量级。该仪器将应用于广泛的研究，例如，定量放射性标记的DNA， RNA和凝胶分离的蛋白质。CCD相机/图像分析系统将允许对目前通过显微显微镜进行定性收集的数据进行定量分析。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，而成本仅为其一小部分。软件控制的步进电机可用于从顺序光学平面获取图像，有效地对标本进行光学切片。每个场的数字化允许对该场中的荧光标记进行定量分析，与光学切片相结合，在标本内预定体积内进行定量。该设施将用于酵母的免疫定位研究，不同发育阶段的转基因果蝇，海鞘幼虫，盘基骨细胞，哺乳动物神经细胞和组织，以及体外微管组装的研究。该荧光光谱仪将允许收集定量荧光强度和动力学数据，这些数据目前无法用范德比尔特文理学院现有的设备获得。例如，利用配备有激发和发射偏振器的T格式荧光光谱仪的止流附件，在极化激发下同时获取平行和垂直发射，将允许实时获取新生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以及具有ca++释放通道的调节蛋白的关联动力学。并将微管蛋白转化为微管。

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.