DECONVOLUTION MICROSCOPE

我们申请资金建立一个4d光学显微镜设备。在考察了几种替代方案后，我们选择了一种显微镜，它带有相关的光源、滤光片、电机驱动器，以及一台计算机、冷却CCD相机和软件包，由科罗拉多州丹佛市的智能成像公司选择和设计。本提案描述了来自五个主要用户和三个次要用户的实验室的科学项目，他们的研究将在很大程度上依赖于该仪器的能力，通过各种荧光团获得的焦点序列对荧光信号进行成像，并从这些数据中反卷积显微镜的离焦传递函数，从而产生更可靠的细胞荧光三维模型。所描述的项目还包括对活细胞的研究。在这里，我们将使用微分干涉对比，和/或标记细胞成分的荧光，在体内跟踪事件。所要求的高效光学系统，连同灵敏的CCD相机，将与具有X，Y，Z控制的机动平台一起使用，以允许对多个细胞进行4-D连续成像，大大提高数据采集速率。特定应用将使用重要的荧光染料、绿色荧光蛋白（GFP）的不同等位基因和/或用不同荧光团标记的纯化蛋白并注射回活细胞。比率成像的能力包括允许特定蛋白质荧光与非特异性细胞标记的正常化，并允许在不同的细胞室离子浓度的测量。该提案描述了仪器技术支持的设施和人员，以及我们在一段时间内对仪器进行管理和维护的计划，该计划远远超出了赠款本身的期限。

We request funds to set up a facility for 4-D light microscopy. After examining several alternatives, we have selected a microscope with associated light sources, filters, and motor drives, together with a computer, cooled CCD camera and software package, selected and designed by Intelligent Imaging of Denver, Colorado. This proposal describes scientific projects from the labs of five major and three minor users whose research will depend heavily on the capabilities of this instrument to image fluorescent signals from through focus series, obtained with a variety of fluorophores, and to deconvolve the out-of- focus transfer function of the microscope from these data, producing more reliable 3-D models of cellular fluorescence. The projects described also include work with live cells. Here we will use differential interference contrast, and/or the fluorescence of tagged cellular components, to follow event in vivo. The efficient optical train requested, together with the sensitive CCD camera, will be used in conjunction with a motorized stage with X,Y,Z control to allow 4-D imaging of multiple cells seriatim, greatly increasing the rate of data acquisition. Specific applications will use vital fluorescent dyes, different alleles of the green fluorescent protein (GFP), and/or purified proteins labeled with different fluorophores and injected back into living cells. A capability for ratio imaging is included to allow both normalization of specific protein fluorescence with non-specific cellular labeling and to permit measurement of ion concentrations in different cellular compartments. The proposal describes facilities and personnel for technical support of the instrument and our plans for instrument management and maintenance over a period extending far beyond the duration of the grant itself.