Live Cell Imaging Confocal Microscope with Spectral Detector and Resonant Scanner

描述(由申请人提供)：这是一项在新泽西医学院成像核心设施内实施先进的活细胞、光谱成像、共振扫描激光共聚焦显微镜的提案。该显微镜将被用来提高研究人员在细胞水平上分析发病机制的能力，以开发预防和阻止疾病进展的手段。主要使用者正在研究的疾病包括艾滋病毒/艾滋病(1例)、白血病(2例)、肺结核(3，4例)、围产期缺氧性脑损伤(5例)、心力衰竭(5例，6，7，8，9例)、酒精性肝病(10，11例)和弓形虫病(12，13例)。初级使用者将利用显微镜研究一种重要的儿科病原体金黄色葡萄球菌、肉毒杆菌毒素、中枢神经系统电极植入的反应、胃肠道辐射损伤和骨质疏松。我们要求的尼康A1Rsi共聚焦显微镜能够提供荧光分子的详细动态和静态图像、这些分子的协调运动、适当标记的细胞内分子彼此之间和与细胞器的相互作用，并将分子变化与细胞的异常行为联系起来。主要用户的几个项目将在活细胞中使用荧光融合蛋白；其他研究人员将使用荧光传感器来监测活细胞内pH、Ca++、cAMP或氧化还原电位的变化，还有一些项目将使用荧光抗体或荧光化学探针来专门定位固定细胞中的表面、细胞质、细胞器和核分子。所要求的显微镜将增强荧光分子在三维中的定位，并增加跟踪活细胞中随着时间推移的荧光分子的能力，并利用多个激光和高分辨率荧光发射光谱在多标记样品中识别特定的荧光分子。软件可用于需要分子缔合测量(FRET)的实验，以及在使用检流计扫描仪进行光激活后利用共振扫描仪动态收集探针的荧光强度。电机驱动平台和图像拼接的组合允许在更大的区域内监控细胞，而不是使用具有高N.A.物镜的单帧图像集合。目前可用的具有2个激光器和2个探测器通道的LSM510不能提供足够的能力来满足本申请中确定的用户或核心设施所服务的其他用户的需求。根据蔡司微成像公司的说法，由于LSM510的配置和陈旧，既不可能升级硬件，也不可能升级软件，也不可能签订服务合同，从而使仪器面临更长的停机时间和可能的全面损失。

DESCRIPTION (provided by applicant): This is a proposal to implement an advanced live cell, spectral imaging, resonant scanning laser confocal microscope within the Imaging Core Facility at New Jersey Medical School. The microscope will be used to enhance the capabilities of researchers to analyze pathogenesis at the cellular level in order to develop means of prevention and of blocking disease progression. Diseases under study by major users of the confocal include HIV-AIDS (1), leukemia (2), tuberculosis( 3,4), perinatal hypoxic brain injury(5), heart failure(5,6,7,8,9), alcoholic liver disease(10,11) and toxoplasmosis(12,13). Minor users will utilize the microscope for their studies of an important pediatric pathogen, K. kingae, botulinum toxin, the reaction to electrode implantation in the CNS, gastrointestinal radiation injury, and osteoporosis. The Nikon A1Rsi confocal microscope we are requesting has the ability to provide detailed dynamic as well as static images of fluorescent molecules, the coordinated movement of such molecules, the interaction of appropriately labeled intracellular molecules with one another and with cell organelles, and to associate molecular changes with deviant behavior of cells. Several of the projects of major users will employ fluorescent fusion proteins in live cells; other investigators will utilize fluorescent sensors to monitor changes in intracellular pH, Ca++, cAMP or redox potential in living cells, and yet others will employ fluorescent antibodies or fluorescent chemical probes to specifically locate surface, cytoplasmic, organelle and nuclear molecules in fixed cells. The requested microscope will enhance localization of fluorescent molecules in 3-dimensions and add the ability to track fluorescent molecules over time in live cells and to identify specific fluorescent molecules in multi-labeled samples utilizing multiple lasers and high resolution fluorescent emission spectra. Software is available for experiments requiring measures of molecular association (FRET) and for kinetic collection of fluorescent intensities of probes with the resonant scanner following photoactivation using the galvanometer scanner. The combination of a motor driven stage and stitching of images allows monitoring of cells over greater areas than possible with single frame collection of images with high N.A. objectives. The currently available LSM510 with 2 lasers and 2 detector channels is not able to provide sufficient capability to meet the needs of neither users identified in this application nor others served by the core facility. Because of the configuration and vintage of the LSM510, according to Zeiss Microimaging, it is not possible to upgrade either the hardware or the software, nor is a service contract available, subjecting the instrument to extended downtime and possible total loss.