Reflected Beam Illumination Microscopy using a Microfluidics Device

描述(由申请人提供)：我们建议建造和使用定制设计的微流控芯片，当与相关的光学元件相结合时，将极大地促进多种形式的荧光超分辨率成像。其基本概念是存在一个反射的平面，使其与盖子滑动成接近45度的法线角度。该反射表面将允许单一成像物镜还在放置在哺乳动物细胞内的任何位置的图像焦平面上提供平面照明。由于只照亮成像的焦平面，大大减少了荧光背景，提高了荧光团的寿命。芯片的单元可访问体积的尺寸将被设计为使单元始终位于可由反射光成像的区域。我们将重点介绍三种成像方式：3D单分子超分辨率(SM-SR)、扩展时间序列、3D成像和3D结构照明显微镜(SIM)。 该项目将围绕微流控装置和光学装置的设计，并评估上述三种成像方式的成像性能。对于每一种方法，我们都将在空间和时间分辨率以及探测器的光稳定性方面将使用该设备时的结果与传统方法进行比较。开发的设备和技术将立即用于研究RBL-2H3细胞中与FceR1相关的信号转导，这是新墨西哥州细胞信号时空建模中心(STMC)的中心焦点。STMC是由NIH资助的系统生物学中心，两位高级人员都是该中心的成员。

DESCRIPTION (provided by applicant): We propose to build and use custom designed microfluidics chips that, when combined with associated optics, will greatly facilitate several modalities of fluorescence super- resolution imaging. The essential concept is the presence of a reflective, planar surface that makes an angle of near 45 degrees with respect to the cover slip normal. This reflective surface will allow a single imaging objective to also provide planar illumination at an image focal plane placed anywhere inside a mammalian cell. Since only the imaged focal plane is illuminated, the fluorescence background is greatly reduced and fluorophore longevity is improved. The dimensions of the cell accessible volumes of the chip will be designed such that cells will always locate to regions amenable for imaging by the reflected light. We will focus on three imaging modalities: 3D Single-Molecule Super-Resolution (SM-SR), extended time series, 3D imaging, and 3D Structured Illumination Microscopy (SIM). The project will focus around design of the microfluidics device and optics, and assessing the imaging performance of each of the three above mentioned imaging modalities. For each, we will compare results when using the device to conventional methods in terms of resolution, both spatial and temporal, and probe photo-stability. The developed devices and techniques will be immediately used for studying FceR1 associated signal transduction in RBL-2H3 cells, which is the central focus of the 'The New Mexico Center for Spatiotemporal Modeling of Cellular Signaling' (STMC). The STMC is an NIH funded center for systems biology, of which both senior personnel are members.