Reflected Beam Illumination Microscopy using a Microfluidics Device

使用微流体装置的反射光束照明显微镜

• 批准号: 8531294
• 负责人: Keith A Lidke
• 金额: $ 18.26万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8531294
• 关键词: Affect; Buffers; Cell model; Cells; Custom; Devices; Dimensions; Dyes; Environment; Equipment; Fluorescence; Funding; Human Resources; Image; Individual; Knowledge; Lateral; Life; Light; Lighting; Longevity; Mammalian Cell; Methods; Microfluidic Microchips; Microfluidics; Microscope; Microscopy; Modality; Modeling; New Mexico; Optics; Oxygen; Pattern; Performance; Production; Proteins; Research Personnel; Resolution; Scanning; Series; Signal Transduction; Speed; Structure; Surface; Systems Biology; Techniques; Technology; Three-Dimensional Imaging; Time; United States National Institutes of Health; cell dimension; cost; design; fluorescence imaging; fluorophore; imaging modality; improved; member; single molecule; spatiotemporal; three dimensional structure

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.

描述（由申请人提供）：我们建议构建和使用定制设计的微流体芯片，当与相关光学器件结合时，将极大地促进荧光超分辨率成像的多种模式。基本概念是存在一个反射性平面，该表面相对于盖玻片法线形成接近 45 度的角度。该反射表面将允许单个成像物镜在位于哺乳动物细胞内任何位置的图像焦平面处提供平面照明。由于仅照亮成像焦平面，因此大大减少了荧光背景并提高了荧光团寿命。芯片的细胞可访问体积的尺寸将被设计为使得细胞总是位于适合反射光成像的区域。我们将重点关注三种成像模式：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装置和光学器件的设计，并评估上述三种成像方式的成像性能。对于每种方法，我们将在空间和时间分辨率以及探测光稳定性方面比较使用该设备与传统方法的结果。开发的设备和技术将立即用于研究 RBL-2H3 细胞中 FceR1 相关信号转导，这是“新墨西哥细胞信号时空建模中心”(STMC) 的核心焦点。 STMC 是 NIH 资助的系统生物学中心，两名高级人员都是该中心的成员。