NANO-RULERS BASED ON SUPPORTED HYBRID LIPID BILAYERS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 时间轨迹分布光学显微镜（TTDOM）最近已在RLBL这里开发，以获得亚衍射光学图像。TTDOM不是记录成像的光强度，而是记录与感兴趣的对象碰撞的单个自由扩散分子信标的荧光解离速率和荧光开启时间。 当囊泡通过激光焦点进行光栅扫描时，平均爆发频率和荧光开启时间的变化比荧光强度的变化要急剧得多。TTDOM的分辨率不受衍射的限制，因此可以提取纳米级的距离。 荧光球常被用作共聚焦光学显微镜的校准和对准的标准样品。 在TTDOM中，荧光解离速率和荧光开启时间的平均值被测量，而不是测量荧光强度，因此需要纳米标尺而不是荧光球来校准显微镜。 这些纳米尺可以是成对或成组的物体，它们的大小、形状和间距都不同。 这些精心设计的图案对应该能够使分子信标改变它们的荧光量子产率或波长，当信标与它们碰撞时，因此在碰撞期间产生荧光猝发。 这些纳米标尺无疑将帮助我们了解TTDOM的能力。 通过在玻璃表面沉积大量的囊泡，然后用缓冲溶液洗涤，逐渐除去一些囊泡，可以得到大小约为50 nm，边缘与边缘最小间距约为130 nm的囊泡对。这不是制备成对囊泡的可控方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Time trajectory distribution optical microscopy (TTDOM) has been recently developed here at RLBL to obtain sub-diffraction optical images. Instead of recording the light intensity for imaging, TTDOM records the fluorescence off-rate and fluorescence on-time of individual freely diffusing molecular beacons colliding with the objects of interest. The mean burst frequency and fluorescence on-time change much more sharply than does the fluorescence intensity when a vesicle is raster scanned through the laser focus. The resolution of TTDOM is not limited by diffraction so nanometer distances can be extracted. Fluorescent spheres are often used as standard samples to calibrate and align the confocal optical microscope. In TTDOM, instead of measuring fluorescence intensity, the mean of fluorescence off-rate and fluorescence on-times are measured, so nanometer rulers rather than fluorescence spheres are needed to calibrate the microscope. Those nano-rulers can be pairs or groups of objects with variable size, shape and separation between them. Those well-engineered patterned pairs should be able to make molecular beacons change their fluorescence quantum yield or wavelength when beacons collide with them and hence generate the fluorescence bursts during collisions. Those nano-rulers will undoubtedly help us to understand the capability of TTDOM. Pairs of vesicles with size of ~50 nm and with minimum separation of ~130 nm from edge to edge have been found. Such pairs were prepared by first depositing a large number of vesicles on the glass surface then gradually removing some from the surface by washing with buffer solution. This is not a controllable way to prepare pairs of vesicles.