To study the behavior of single biomolecules in the live cell environment, a method is required which is able to access the temporal (microsecond) and spatial (nanometer) scales relevant to single molecules while still being able to image the larger cellular context in three dimensions. To answer this challenge, we have developed 3D Contextualized Real-time Tracking (3D-CRT) microscopy, which combines real-time 3D single-particle tracking with the larger-scale contextual information provided by two-photon laser scanning microscopy (2P-LSM). Real-time tracking is performed using a high-speed 3D piezoelectric stage and fast feedback to lock a freely moving nanoscale probe in the focus of the microscope's objective. The 2P-LSM module, slaved by the moving nanoprobe, collects optical sections to build up a large-scale 3D volumetric image of the environment. Using HIV-TAT modified nanoparticles as a model for the HIV-1 virus, we utilize 3D-CRT microscopy to observe the transient landing events of freely diffusing nanoparticles on the 3D cell surface (Fig. 1). Furthermore, the 3D-CRT microscope is able to map local membrane dynamics in three dimensions due to its high time resolution (10 �sec), while the high localization precision (down to 10 nm) enables resolution of the complex 3D structures of macropinosomes and filopodia in live cells.

3D contextualized real-time tracking microscopy for the elucidation of 3D structure and dynamics in live cells

246th National Meeting (2013)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.