Reflected Beam Illumination Microscopy using a Microfluidics Device

描述（由申请人提供）：我们建议建立和使用定制设计的微流体芯片，当与相关光学相结合时，将极大地促进荧光超分辨率成像的几种模式。基本概念是存在一个反射的平面表面，使其相对于覆盖滑动法线的角度接近45度。这种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成像焦平面上提供平面照明。由于只有成像焦平面被照亮，荧光背景大大减少，荧光团寿命得到改善。芯片的细胞可访问体积的尺寸将被设计成这样，细胞将始终定位到可被反射光成像的区域。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）。

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.