This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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