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

描述(申请人提供)：荧光显微镜具有独特的能力，可以探测静态和实时过程，具有极高的特异性，可以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级的四维(4D)多色活体数据，并具有双重功能来实现亚衍射结构照明(SI) 固定样品的成像。在过去的两年里，加州大学旧金山分校的第一代OMX显微镜已经从专门的开发显微镜转换为生产显微镜，向加州大学旧金山分校内部和外部学术界的项目开放。作为更普遍使用的结果，用户确定了几个主要的期望改进，如果能够对OMX显微镜进行改进，将极大地扩展他们实现研究目标的能力。第一个更新是增加在不受干扰的自然状态下观察生物过程的时间。光毒是活体显微镜的一个主要限制因素，它会导致细胞形态改变、细胞进程延迟和细胞死亡。引入脉冲激光，将激发光减少到微秒曝光，而不是目前的毫秒限制，将允许实现更低的光子剂量，使细胞能够进行更长时间的成像。第二个更新是利用相机和舞台技术的最新改进，提高3D结构照明数据采集的速度和稳定性。这 更新将有一个额外的好处，即允许在体内进行3D SI。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。在这项应用中，我们寻求资金来革新OMX显微镜的技术基础，以实现更快、更稳定的实时和SI成像数据采集。建议的改进包括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)采用更快的采集速率的sCMOS相机，与脉冲激光相结合，使3D SI数据堆栈能够在3秒内采集到，而不是目前的13分钟；3)用更现代的平台取代目前引入热致漂移的XYZ平台，以提高SI数据采集的稳定性、速度和深度；以及4)将控制新平台电机的计算机升级到带有PCI/PCI(E)插槽的平台。这些技术进步将使许多由美国国立卫生研究院资助的生物医学研究项目受益，并将对阐明许多人类疾病背后的基本生物学过程至关重要。 与公共卫生相关：研究在非扰动环境下发生在活体中的动态过程对于理解导致许多人类疾病的生物过程的基本机制至关重要。快速三维在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有力的新技术。这一应用将1)极大地扩展这项技术的能力，使其在生物过程的整个过程中不受其自然状态的干扰，2)实现对活样本的超分辨率显微镜，此前只能在非活样本中进行检查。

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.