Reflected Beam Illumination Microscopy using a Microfluidics Device

描述（由申请人提供）：我们提出构建和使用定制设计的微流体芯片，当与相关光学器件组合时，其将极大地促进荧光超分辨率成像的几种模态。基本概念是存在相对于盖玻片法线成近45度角的反射性平坦表面。该反射表面将允许单个成像物镜也在放置在哺乳动物细胞内任何地方的图像焦平面处提供平面照明。由于只有成像的焦平面被照亮，荧光背景大大减少，荧光团的寿命得到改善。芯片的细胞可接近体积的尺寸将被设计成使得细胞将总是位于适于通过反射光成像的区域。我们将专注于三种成像模式：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）。 该项目将重点关注微流体设备和光学器件的设计，并评估上述三种成像方式的成像性能。对于每一个，我们将比较结果时，使用该设备的传统方法的分辨率，空间和时间，探测光稳定性。开发的设备和技术将立即用于研究RBL-2 H3细胞中FceR 1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NIH资助的系统生物学中心，两名高级人员都是其成员。

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.