Biophotonics: Multicolor total internal reflection fluorescence microscopy

一些最顽固而又耐人寻味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，同时保持一种渗透屏障，以阻止营养物质进入和毒素进入。细胞膜是细胞分泌自己的信号并检测邻居信号的地方。膜不仅是动态生理学的场所，也是病理学的场所。神经系统的许多疾病影响细胞在细胞膜上分泌或检测信号的能力。对于许多病原体来说，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，它们也需要保持膜的完整性，这样我们的细胞才能在适宜生存的环境中生存。细胞膜厚度为4 nm。因此，光的波长限制了我们在来自细胞内部的信号的背景中检测膜的直接环境中发生的变化的能力。全内反射荧光显微镜(TIF-FM)是一种将激发限制在盖片上方50-70 nm平面内的技术，已成功地用于成像发生在质膜附近的这一空间内的事件。这项提议将从三个方面扩展TIR-FM的能力：1)允许同时跟踪多个荧光团；2)允许通过TIF-FM同时研究细胞表面事件和通过表观荧光研究内部变化；3)允许在使用TIR-FM的同时通过紫外光对信号进行局部光激活。

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.