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Upgrading the OMX microscope for extended live imaging and fast live 3-D structur

升级 OMX 显微镜以实现扩展实时成像和快速实时 3D 结构

基本信息

批准号：
8246972
负责人：
JENNIFER C FUNG
金额：
$ 17.76万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-15 至 2013-05-14
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Fluorescence microscopy has the unique capacity to probe both static and live processes with great specificity to link the dynamics and/or localization of molecular and cellular components with their function. Recently, a new microscope platform, OMX, was designed to acquire sub-second four-dimensional (4D) multi-color in vivo data with a dual functionality to attain sub-diffraction structured illumination (SI) imaging of fixed samples. Over the last two years, this first generation OMX microscope at UCSF has been converted from a dedicated development microscope to a production microscope open to projects from within UCSF and from the outside academic community. As a result of more general use, users identified several major desired improvements which, if they could be made to the OMX microscope, would vastly expand their ability to attain their research goals. The first update is to increase the time over which biological processes can be observed in their unperturbed natural state. Phototoxicity is a major limitation in live microscopy, inducin morphological changes, delays in cell progression and cell death. Introduction of pulsed lasers to reduce the excitation light to microsecond exposures rather than the current millisecond limits will permit far lower photon doses to be achieved, allowing cells to be imaged for much longer periods of time. The second update is to use recent improvements in camera and stage technology to increase the speed and stability of 3D structural illumination data acquisition. This update will have the added benefit of permitting in vivo 3D SI. Currently the quality and throughput of 3D SI microscopy is severely compromised by drift between consecutive sections in a 3D image stack. In this application, we seek funds to revolutionize the technological base of the OMX microscope for far faster and more stable data acquisition for both live and SI imaging. The proposed enhancements include 1) upgrading our lasers to pulsed lasers to achieve microsecond exposure times; 2) incorporating a sCMOS camera with faster acquisition rates, which in combination with the pulsed lasers will permit a 3D SI data stack to be acquired in 3 seconds rather than the current 13 minutes; 3) replacing our current xyz stage that introduces thermally induced drifts with a more modern stage to improve the stability, speed and depth of SI data acquisitions; and 4) upgrading the computer that will control the new stage motors to one with PCI/PCI(e) slots. These technological advances will benefit a great many biomedical research projects funded by NIH and will be of vital importance in elucidating the basic biological processes underlying many human diseases. PUBLIC HEALTH RELEVANCE: Studies of the dynamic processes occurring in living organisms in a non-perturbed setting is of vital importance in understanding the basic mechanisms of the biological processes underlying many human diseases. Rapid three-dimensional in vivo and super-resolution structured illumination imaging have become powerful new techniques in monitoring the changes that occur in the cell. This application will 1) greatly extend the ability of this technology to follow a biological process through its entire course without perturbation of its natural state and 2) enable super-resolution microscopy on live samples heretofore could only be examined in non-living specimens.

描述（由申请人提供）：荧光显微镜具有独特的能力，能够以极高的特异性探测静态和活过程，将分子和细胞成分的动态和/或定位与其功能联系起来。最近，一种新的显微镜平台，OMX，被设计用于获取亚秒四维（4D）多色体内数据，具有双重功能，以获得亚衍射结构照明（SI）固定样品的成像。在过去的两年中，UCSF的第一代OMX显微镜已经从专用的开发显微镜转换为生产显微镜，对UCSF内部和外部学术界的项目开放。由于更普遍的使用，用户确定了几个主要的改进，如果他们可以对OMX显微镜，将大大扩大他们的能力，以实现他们的研究目标。第一个更新是增加生物过程在未受干扰的自然状态下可以观察到的时间。光毒性是活体显微镜检查的主要限制，诱导形态学变化，延迟细胞进展和细胞死亡。引入脉冲激光器将激发光的曝光时间减少到微秒，而不是目前的毫秒限制，这将允许实现更低的光子剂量，从而使细胞能够在更长的时间内成像。第二个更新是使用相机和载物台技术的最新改进，以提高3D结构照明数据采集的速度和稳定性。这更新将具有允许体内3D SI的额外益处。目前，3D SI显微镜的质量和吞吐量受到3D图像堆栈中连续切片之间的漂移的严重影响。在此应用中，我们寻求资金来彻底改变OMX显微镜的技术基础，以便更快，更稳定地采集实时和SI成像的数据。建议的增强功能包括：1）将我们的激光器升级为脉冲激光器，以实现微秒级曝光时间; 2）采用具有更快采集速率的sCMOS相机，与脉冲激光器相结合，将允许在3秒内采集3D SI数据堆栈，而不是目前的13分钟; 3）用更现代的阶段取代我们目前引入热致漂移的xyz阶段，以提高SI数据采集的稳定性、速度和深度;以及4）将控制新载物台电机的计算机升级为具有PCI/PCI（e）插槽的计算机。这些技术进步将使NIH资助的许多生物医学研究项目受益，并将对阐明许多人类疾病的基本生物学过程至关重要。公共卫生相关性：在非扰动环境中研究生物体中发生的动态过程对于理解许多人类疾病的生物过程的基本机制至关重要。快速三维活体成像和超分辨率结构照明成像已成为监测细胞变化的强大新技术。该应用将1）极大地扩展该技术在其整个过程中跟踪生物过程而不干扰其自然状态的能力，以及2）使超分辨率显微镜能够在活体样品上进行迄今为止只能在非活体标本中进行检查。