Biophotonics: Multicolor total internal reflection fluorescence microscopy

生物光子学：多色全内反射荧光显微镜

• 批准号: 0322867
• 负责人: Sanford Simon
• 金额: --
• 依托单位: Rockefeller University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0322867&HistoricalAwards=false
• 关键词: Biophotonics; Multicolor; total; internal; reflection

0322867SimonSome of the most intransigent, yet intriguing biological questions occur at cellular membranes. At the membrane the cell has to take up select substrates while maintaining a permeability barrier that keeps nutrients in and toxins out. The membrane is the site where cells secrete their own signals and detect those of their neighbors. The membrane is not only the site of a dynamic physiology but pathology as well. Many diseases of the nervous system affect either the ability of cells to secrete or detect signals at the cell membrane. For many pathogens our cells are a tempting home. They need to compromise the membranes to enter. However, they also need to maintain membrane integrity so our cells can survive as a hospitable environment. The cell membrane is 4 nm thick. Thus, the wavelength of light constrains our ability to detect changes occurring in the immediate environment of the membrane in the background of signals from the interior of the cell. Total internal reflection fluorescence microscopy (TIF-FM) is a technique that limits the excitation to a plane of 50-70 nm above a coverslip and has been successfully used to image events occurring within this space adjacent to the plasma membrane. This proposal will extend the capabilities of TIR-FM in three ways: 1) Allow multiple fluorophores to be followed simultaneously; 2) Allow simultaneous study of cell surface events by TIF-FM and internal changes by epi-fluorescence; 3) Allow localized photoactivation of signaling by UV while using TIR-FM.

[0322867]西蒙：一些最难以妥协，但又最有趣的生物学问题发生在细胞膜上。在细胞膜上，细胞必须吸收选定的底物，同时保持一个渗透性屏障，使营养物质进入，毒素排出。细胞膜是细胞分泌自身信号并检测邻近细胞信号的地方。膜不仅是动态生理的场所，也是病理的场所。许多神经系统疾病影响细胞分泌或检测细胞膜信号的能力。对许多病原体来说，我们的细胞是一个诱人的家园。它们需要破坏细胞膜才能进入。然而，它们也需要保持细胞膜的完整性，这样我们的细胞才能在适宜的环境中生存。细胞膜厚度为4nm。因此，光的波长限制了我们在细胞内部信号的背景下检测膜的直接环境中发生变化的能力。全内反射荧光显微镜（tiff - fm）是一种将激发限制在盖盖上方50-70 nm的平面上的技术，已经成功地用于成像发生在靠近质膜的这个空间内的事件。该提案将从三个方面扩展TIR-FM的功能：1)允许同时跟踪多个荧光团；2)允许同时用TIF-FM研究细胞表面事件和用外显荧光研究细胞内部变化；3)在使用TIR-FM时，允许紫外线局部光激活信号。