Assessing Location and Distribution of Proteins at the Nuclear Envelope from Dual Color Z-Scan Intensity Profiles

The nuclear envelope (NE) consists of the outer membrane (ONM) and inner (INM), which are concentric separated by tens nanometers. This structure separates nucleus from cytoplasm hosts various proteins that essential for a variety signaling processes. NE divided into INM ONM proteins, reflecting their preferential location as dictated function. However, live-cell determination distribution across both membranes has been experimentally challenging due to extreme close proximity membranes. Here, we utilize knowledge point spread function two-photon microscope extract fluorescently tagged proteins. We do this performing axial scans cell expressing two labeled with different colors. One protein serves reference marker, while other is interest. dual-color z-scan approach determines relative separation an accuracy few nanometer. verified capabilities technique using in vitro engineered systems vivo model systems. In addition, used create locality map check internal consistency technique. same further determined thickness INM. Finally, perform time-resolved measurements explore transport real time. work supported grants National Institutes Health (R01 GM064589, R01 GM098550, RO1 GM124279, GM129374).