NANO-RULERS BASED ON SUPPORTED HYBRID LIPID BILAYERS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 时间轨迹分布光学显微镜(TTDOM)是最近在这里发展起来的，用于获得亚衍射光学图像。TTDOM不是记录用于成像的光强度，而是记录单个自由扩散的分子信标与感兴趣对象碰撞时的荧光关闭速率和荧光开启时间。当水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剧烈得多。TTDOM的分辨率不受衍射的限制，因此可以提取纳米距离。 荧光微球常被用作校准和对准共焦光学显微镜的标准样品。在热释光显微镜中，不是测量荧光强度，而是测量荧光偏移率和荧光开启时间的平均值，因此需要用纳米尺子而不是荧光小球来校准显微镜。这些纳米尺子可以是成对或成组的物体，它们之间的大小、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在信标与其碰撞时改变其荧光量子产率或波长，从而在碰撞过程中产生荧光脉冲。这些纳米尺子无疑将有助于我们了解TTDOM的能力。已发现大小为~50 nm的囊泡对，从边到边的最小间隔为~130 nm。这种对是通过首先在玻璃表面沉积大量小泡，然后通过缓冲液清洗逐渐从表面去除一些来制备的。这不是一种可控的方法来制备成对的囊泡。

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.