Histology, Microscopy and Imaging

组织学、显微镜和成像

• 批准号: 8734427
• 负责人: Irina A Pikuleva
• 金额: $ 8.66万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734427
• 关键词: Ablation; Animals; Area; Automation; Biomedical Research; Calendar; Cancer Center Support Grant; Carbon Dioxide; Cell Count; Cell Culture Techniques; Cell Survival; Cells; Center Core Grants; Charge; Collaborations; Color; Computer software; Computers; Consult; Consultations; Coupled; DNA analysis; Dermatology; Dissection; Doctor of Philosophy; Due Process; Electron Microscope; Electron Microscopy; Embryo; Engineering; Equipment; Experimental Designs; Eye; Fee-for-Service Plans; Floor; Fluorescence; Fluorochrome; Freezing; Funding; Genetic; Grant; Hematoxylin and Eosin Staining Method; Histology; Hour; Housing; Image; Image Analysis; Immunohistochemistry; Incubators; Individual; Industry; Institutes; Journals; Laboratories; Laboratory Research; Lasers; Letters; Licensing; Life; Light; Maintenance; Malignant Neoplasms; Measurement; Measures; Methods; Microscope; Microscopy; Microtome - medical device; Monitor; Motor; Occupations; Oils; Optic Nerve; Optics; Paraffin; Pathology; Persons; Phase; Photography; Positioning Attribute; Preparation; Procedures; Process; Production; Protein Analysis; Proteomics; Publications; RNA analysis; Research Personnel; Resources; Role; Running; Sales; Sampling; Scanning; Science; Services; Slide; Specific qualifier value; Specimen; Spottings; Staging; Staining method; Stains; System; Techniques; Temperature; Time; Tissues; Training; Trichrome stain method; United States National Institutes of Health; Vision; Vision research; Work; Writing; Zebrafish; base; cell injury; cellular imaging; charge coupled device camera; cryostat; density; design; detector; digital; digital imaging; experience; flexibility; instrument; instrumentation; investigator training; laser capture microdissection; medical schools; meetings; member; ranpirnase; repaired; research study; tissue culture; tissue fixing; tissue processing; tissue/cell culture; transmission process; web site

Module 2. Histology, Microscopy and Imaging (HMI) Eric Pearlman, Ph.D., Director, Catherine Doller, Histology Manager, Scott J. Howell, Ph.D., Microscopy and Digital Imaging Manager Overview and Resources: In the previous funding period, Histology was administratively part of the Tissue Culture and Cell Analysis Module; however, due to changes in investigator needs, increased microscopy and imaging capacity, we have now combined Histology with Imaging and added Microscopy. The Histology, Microscopy and Imaging (HMI) Module will occupy two rooms in the consolidated VSRC Core Grant facility in the Institute of Pathology, with a third room in Dr. Pearlman's laboratory containing animal photography equipment. Catherine Doller will continue as the Histology Manager, and Scott Howell, Ph.D. will manage the Microscopy and Imaging functions of the HMI Module. Histology Function The Histology function of this Module has been fully set up for processing and sectioning paraffin and frozen tissues since the initiation of the Core Grant supported facilities in 1997. A detailed description of the equipment is listed on the Resources page (Form HH). The major purchase since the previous submission is a new cryostat to replace the one utilized since 1997 (using P30 supplemental funds). All other equipment, including the tissue processor and microtome, remain in excellent working order. Tissue Processing and Sectioning The Histology Module Manager provides service for cryostat and microtome sections. Ms Doller's experience enables her to dissect and orient ocular tissues to obtain the optimal sections. Examples include repeat sections through the optic nerve and in embryonic zebrafish eyes. Staining techniques include basic hematoxylin and eosin, PASH and Massons Trichrome. Although we included immunohistochemistry in the past, the increased volume of tissues to be processed, sectioned and stained precludes our ability to offer this service in the next cycle. However, Ms Doller is well experienced in this area and will continue to advise investigators on procedures. Microscopy and Imaging Functions Dr. Scott Howell has been managing the microscopy and imaging functions in the VSRC since 2006. He oversees the equipment in the main core lab in the Path Institute, and has access to several microscopes in other departments in the School of Medicine, and advises on the use of electron microscopy in the Department of Material Sciences. Available microscopes include: In the Institute of Pathology room 106: Leica Live Cell Imaging System: The most heavily used microscope is a Leica DM1 6000B automated inverted microscope with fluorescent and phase capable optics (Details and images are on the website, http://www.case.edu/med/vsrc/). Objectives on this microscope include a 10x, 20x, 40x and 63x high NA oil. Images are acquired on this system by a Retiga Exi BW/Color camera. Due to its automation and associated software this microscope can function as a multidimensional acquisition (MDA) station. For example, an experiment can be devised that would allow a researcher to scan each well of a 96 well plate at, for example, 3 spots in each well. At any position, a fluorescent image (using 1, 2 or 3 different fluorochromes) and an accompanying phase contrast image can be acquired. Z stacks can also be acquired at any of the positions. The incubator allows 37 º C and 5% C02 to be maintained for an indefinite time period allowing for the planning of multi day time-lapse experiments as needed. The ability to conduct MDA experiments allows VSRC investigators to examine the cells in real time. Another feature of the automated stage is the capacity to stitch together a sequence of high magnification images into one single image. Since the microscope has a 63x high NA oil objective and a precisely controlled Z-motor, researchers can generate 'confocal' like stacks in less time than it would normally take on a laser scanning confocal microscope, thus protecting the live cells from the damaging effects of the laser. Although these images are not as "sharp" as those generated by a laser scanning confocal, once they are processed via deconvolution software, the image quality rivals that of a laser scanning confocal. Presently the HMI Module has unlimited access to Velocity deconvolution software in the Department of Genetics in the adjacent Biomedical Research Building. The HMI Module also has a copy of Autoquant deconvolution software. The automation of this microscope, stage and camera are all controlled by Metamorph Premier Software. Sign-up for usage of the live cell microscope is on-line through the VSRC web site, and VSRC investigators will contact Dr.Howell to image samples directly, or for training on this instrument. Olympus BX-60 Upright Microscope: This microscope is used for routine examination of slides and for fluorescence. The microscope is housed in the new VSRC Core Grant facility in the Institute of Pathology. This scope has DIG and fluorescent capabilities with up to 100x high NA oil objectives. The microscope is coupled to a Q imaging EXi Aqua Blue camera and is capable of BW and Color imaging. This microscope has been very heavily utilized and access is by an on-line sign up calendar on the VSRC web site. VSRC investigators will contact Dr.Howell for training. Metamorph Premier Offline Imaging Software: This industry standard software allows for a wide range of image analysis including but not limited to basic measuring, cell counting, density measurements and the writing of journals to allow for automated image analysis. Five licenses of this software have been purchased to allow for image analysis to occur not only in the confines of the Module, but also in the individual research laboratories. The Module's copy of software is run on a T3500 Dell Computer with 12GB of RAM to provide the computational power needed for image analysis. Additional Instruments used by VSRC investigators Spinning disc Confocal microscope: The Department of Dermatology in the BRB 5th floor purchased a Perkin Elmer Ultraview VoX Spinning Disc Confocal Microscope in 2009. Dr.Howell was intimately involved in the purchasing process due to his previous experience in microscopy sales. Since then Dr.Howell has been the chief trainer and contact person along with Dr.Minh Lam of Dermatology. This unique situation grants VSRC members priority access to this confocal microscope. This particular microscope was purchased mainly to conduct live cell imaging experiments. It is equipped with a temperature control chamber to maintain a constant 37ºC and also a CO2 delivery system to conduct, if needed, multi day experiments. The system also has a motorized stage to allow multipoint acquisitions over time. Four lasers are currently installed on the system and include 405nm, 488nm, 561 nm and a 640nm wavelengths, allowing researchers flexibility in probe choices. When compared to classic laser scanning confocal microscopes the spinning disc can capture the entire field of view at once which is in contrast to the laser scanning confocal which captures the image in a point by point fashion. Because of this design, the spinning disc has a much greater acquisition rate. Detectors on the spinning disc confocal make use of CCD cameras and are extremely sensitive to low levels of light, which allows for quicker exposure times. Therefore the spinning disc confocal is the best choice for live cell applications, since the lower light exposure maintains cell viability. The spinning disc has been extensively used by VSRC investigators including Drs. Kern, Park, Pearlman, Pikuleva and Iyengar. Laser Capture Microdissection: LCM can isolate specific cells from either tissue or cell culture for RNA, DNA or protein analysis, and sufficient material can be obtained for analysis by qPCR, microarray or proteomics. This LCM has a UV cutting laser, which is used for dissection or for ablation of unwanted material, while the infrared laser is used for specimen capture. The LCM was acquired by a NIH shared instrumentation grant that included VSRC investigators with the support of the School of Medicine, and is administered by the Case Comprehensive Cancer. This instrument has been used by Drs. Kern, Lin, Palczewski, Pearlman and Pikuleva. Electron Microscopy - TEM. SEM; Although not an integral part of the VSRC core. Dr. Howell assists investigators to utilize electron microscopes in the Department of Pathology (in the Institute) for tissue processing and microscopy. However, state of the art microscopes are located in the nearby Department of Materials Sciences and Engineering, which include three 3 scanning electron microscopes and 3 transmission electron microscopes http://dmseg5.case.edu/Groups/Ernst/scsam.html. Microscope use is on a fee for service basis, and has been and will continue to be utilized by Drs. Kern, Palczewski and Pearlman. [see Engel and Palczewski J Neur Methods 2011, and the letter of support from Dr Heuer]

模块 2. 组织学、显微镜和成像 (HMI) Eric Pearlman 博士，主任，Catherine Doller，组织学经理，Scott J. Howell 博士，显微镜和数字成像经理 概述和资源： 在上一个资助期间，组织学在管理上是组织培养和细胞分析模块的一部分；然而，由于研究者需求的变化、显微镜和成像能力的提高，我们现在将组织学与成像相结合，并增加了显微镜。组织学、显微镜和成像 (HMI) 模块将占用病理学研究所 VSRC 核心拨款综合设施中的两个房间，第三个房间位于 Pearlman 博士的实验室，配有动物摄影设备。 Catherine Doller 将继续担任组织学经理，Scott Howell 博士将继续担任组织学经理。将管理 HMI 模块的显微镜和成像功能。 组织学功能 自 1997 年核心拨款支持设施启动以来，该模块的组织学功能已完全建立用于处理和切片石蜡和冷冻组织。资源页面（表格 HH）列出了设备的详细说明。自上次提交以来的主要采购是一台新的低温恒温器，以取代自 1997 年以来使用的低温恒温器（使用 P30 补充资金）。所有其他设备，包括组织加工机和切片机，均保持良好的工作状态。 组织处理和切片 组织学模块管理器为低温切片机和切片机切片提供服务。多勒女士的经验使她能够解剖和定向眼组织以获得最佳切片。例子包括通过视神经和胚胎斑马鱼眼睛的重复切片。染色技术包括碱性苏木精和伊红、PASH 和 Massons Trichrome。尽管我们过去包括了免疫组织化学，但待处理、切片和染色的组织量的增加使我们无法在下一个周期提供这项服务。然而，多勒女士在这一领域经验丰富，并将继续就程序向调查人员提供建议。 显微镜和成像功能 Scott Howell 博士自 2006 年以来一直管理 VSRC 的显微镜和成像功能。他负责监督 Path 研究所主要核心实验室的设备，并可以使用医学院其他部门的几台显微镜，并就材料科学系电子显微镜的使用提供建议。可用的显微镜​​包括： 病理研究所 106 室：Leica 活细胞成像系统：使用最频繁的显微镜是 Leica DM1 6000B 自动倒置显微镜，配有荧光和相位光学器件（详细信息和图像位于网站 http://www.case.edu/med/vsrc/）。该显微镜的物镜包括 10 倍、20 倍、40 倍和 63 倍高数值孔径油。该系统上的图像由 Retiga Exi BW/Color 相机采集。由于其自动化和相关软件，该显微镜可以充当多维采集 (MDA) 站。例如，可以设计允许研究人员扫描96孔板的每个孔的实验，例如每个孔中的3个点。在任何位置，都可以获取荧光图像（使用 1、2 或 3 种不同的荧光染料）和随附的相差图像。 Z 堆栈也可以在任何位置获取。培养箱可以无限期地维持 37°C 和 5% CO2，从而可以根据需要规划多天的延时实验。进行 MDA 实验的能力使 VSRC 研究人员能够实时检查细胞。自动化平台的另一个特点是能够将一系列高放大倍率图像拼接成一个图像。由于该显微镜具有 63 倍高数值孔径油物镜和精确控制的 Z 电机，因此研究人员可以在比激光扫描共焦显微镜正常情况更短的时间内生成类似“共焦”的堆栈，从而保护活细胞免受激光的破坏性影响。尽管这些图像不像激光扫描共焦生成的图像那么“清晰”，但一旦通过反卷积软件处理，图像质量就可以与激光扫描共焦的图像质量相媲美。目前，HMI 模块可以无限制地访问邻近生物医学研究大楼遗传学系的 Velocity 反卷积软件。 HMI 模块还具有 Autoquant 反卷积软件的副本。该显微镜、载物台和相机的自动化均由 Metamorph Premier 软件控制。通过 VSRC 网站在线注册使用活细胞显微镜，VSRC 研究人员将联系 Howell 博士直接对样本进行成像，或接受有关该仪器的培训。 奥林巴斯 BX-60 正置显微镜：该显微镜用于载玻片的常规检查和荧光检查。该显微镜位于病理研究所新的 VSRC Core Grant 设施中。该示波器具有 DIG 和荧光功能，具有高达 100 倍的高 NA 油物镜。该显微镜与 Q 成像 EXi Aqua Blue 相机连接，能够进行黑白和彩色成像。该显微镜已被广泛使用，可通过 VSRC 网站上的在线注册日历进行访问。 VSRC 调查人员将联系 Howell 博士进行培训。 Metamorph Premier 离线成像软件：该行业标准软件可进行广泛的图像分析，包括但不限于基本测量、细胞计数、密度测量和撰写期刊以进行自动图像分析。已购买该软件的五个许可证，以便不仅可以在模块范围内进行图像分析，还可以在各个研究实验室中进行图像分析。该模块的软件副本在具有 12GB RAM 的 T3500 Dell 计算机上运行，​​以提供图像分析所需的计算能力。 VSRC 研究人员使用的其他仪器 转盘共聚焦显微镜：BRB 5 楼皮肤科于 2009 年购买了一台 Perkin Elmer Ultraview VoX 转盘共聚焦显微镜。Howell 博士由于之前从事显微镜销售的经验，因此密切参与了采购过程。从那时起，Howell 医生与皮肤科的 Minh Lam 医生一起担任首席培训师和联系人。这种独特的情况使 VSRC 成员能够优先使用这款共聚焦显微镜。购买这种特殊的显微镜主要是为了进行活细胞成像实验。它配备了一个温度控制室，可保持恒定的 37°C，还配备了二氧化碳输送系统，可在需要时进行多天实验。该系统还具有一个电动平台，可以随着时间的推移进行多点采集。目前该系统上安装了四个激光器，包括 405nm、488nm、561nm 和 640nm 波长，使研究人员能够灵活选择探头。 与传统的激光扫描共焦显微镜相比，旋转盘可以一次捕获整个视野，这与以逐点方式捕获图像的激光扫描共焦显微镜形成鲜明对比。由于这种设计，旋转盘具有更高的采集率。旋转圆盘共焦上的探测器使用 CCD 相机，对低光水平极其敏感，从而可以缩短曝光时间。因此，转盘共焦是活细胞应用的最佳选择，因为较低的光照可以维持细胞活力。 VSRC 研究人员（包括 Drs.） 广泛使用了旋转盘。科恩、帕克、皮尔曼、皮库列娃和艾扬格。 激光捕获显微切割：LCM 可以从组织或细胞培养物中分离特定细胞进行 RNA、DNA 或蛋白质分析，并且可以获得足够的材料用于 qPCR、微阵列或蛋白质组学分析。该 LCM 具有紫外切割激光器，用于解剖或烧蚀不需要的材料，而红外激光器则用于样本捕获。 LCM 是由 NIH 共享仪器拨款获得的，其中包括 VSRC 研究人员在医学院的支持下，并由案例综合癌症中心管理。该仪器已被 Drs 使用。科恩、林、帕尔切夫斯基、皮尔曼和皮库列娃。 电子显微镜 - TEM。扫描电镜；虽然不是 VSRC 核心的组成部分。 Howell 博士协助研究人员利用病理学系（研究所）的电子显微镜进行组织处理和显微镜检查。然而，最先进的显微镜位于附近的材料科学与工程系，其中包括三台 3 扫描电子显微镜和 3 台透射电子显微镜 http://dmseg5.case.edu/Groups/Ernst/scsam.html。显微镜的使用是按服务收费的，并且已经并将继续由博士使用。科恩、帕尔切夫斯基和皮尔曼。 [参见 Engel 和 Palczewski J NeurMethods 2011，以及来自 Dr Heuer 的支持信]