Achieving Axial Super-Resolution With The Two-Photon Dual-Color Z-Scan Method

We developed dual-color (DC) fluorescence z-scans to determine the axial location of proteins inside the living cell with a resolution on the order of ten nanometer. The technique is validated by using engineered systems and subsequently applied to determine the thickness of the nuclear envelope. We further demonstrate the ability to distinguish proteins associated with the inner and outer nuclear membranes. Additionally, we explored the application of the DC z-scan technique for determining the size of sub-diffraction particles immobilized on glass. It is well established by electron microscopy that retroviruses produce particles with significant differences in their diameter. To investigate this structural heterogeneity, virus-like particles (VLPs) were produced by the expression of the fluorescently labeled retroviral structural protein, Gag, in mammalian cells and subsequently immobilized on glass slides. DC z-scans were then used to measure the size of each VLP and their fluorescence intensity, which is proportional to the amount of incorporated labeled Gag. The relationship between fluorescence intensity and particle size provides a unique approach for investigating the mechanism of retroviral assembly, which has been difficult to attain via other techniques. This work is supported by grants from the National Institutes of Health (RO1 GM64589, RO1 GM098550, RO1 GM124279).