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）：荧光显微镜具有具有静态和实时过程的独特能力，具有极大的特异性，以将分子和细胞成分的动力学和/或定位与其功能联系起来。最近，一个新的显微镜平台OMX旨在获取具有双重功能的次秒四维（4D）多色在体内数据中，以获得亚分化结构化照明（SI）固定样品的成像。在过去的两年中，UCSF的第一代OMX显微镜已从专用开发显微镜转换为开放的生产显微镜，转换为UCSF内部和外部学术界的项目。由于使用了更一般的用途，用户确定了几种重大期望的改进，如果可以对OMX显微镜进行这些改进，将大大扩展其实现研究目标的能力。第一个更新是增加可以在其不受干扰的自然状态下观察到的生物过程的时间。光毒性是实时显微镜，诱导素形态变化，细胞进展和细胞死亡的主要局限性。引入脉冲激光器以将激发光降低至微秒暴露，而不是当前的毫秒限制，将允许达到较低的光子剂量，从而可以将细胞成像更长的时间。第二个更新是利用摄像机和舞台技术的最新改进来提高3D结构照明数据获取的速度和稳定性。这更新将带来允许体内3D SI的额外好处。当前，3D SI显微镜的质量和吞吐量因3D图像堆栈中连续部分之间的漂移而严重损害了。在此应用程序中，我们寻求资金来彻底改变OMX显微镜的技术基础，以更快，更稳定的数据获取为实时和SI成像。提出的增强功能包括1）将激光器升级为脉冲激光器以达到微秒的暴露时间； 2）合并具有更快采集率的SCMOS摄像头，与脉冲激光器结合使用，将允许在3秒内而不是当前13分钟内获取3D SI数据堆栈； 3）取代我们当前的XYZ阶段，该阶段以更现代的阶段引入热诱导的漂移，以提高SI数据采集的稳定性，速度和深度； 4）将将新舞台电动机控制在PCI/PCI（E）插槽的计算机上。这些技术进步将使由NIH资助的许多生物医学研究项目受益，对于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：对在非扰动环境中生物中发生的动态过程的研究对于理解许多人类疾病背后的生物学过程的基本机制至关重要。快速的三维体内和超分辨率结构化照明成像已成为监测细胞中发生的变化的强大新技术。该应用程序将1）大大扩展了该技术在整个过程中遵循生物过程的能力，而无需对其自然状态扰动，并且2）仅在非生存标本中检查对现场样本的超分辨率显微镜。