In addition to our use of super-resolution in studies described above, we continue to focus on the interaction of lytic granules with the cytoskeleton. Using STED nanoscopy we visualized individual myosin IIa filaments wrapped around granules, both isolated and within whole cells . These images enabled us to discern between alternative models of myosin IIa-granule interactions suggested by biochemical studies. We also have applied the use of STED to study and define granule size , and have optimized dual channel STED for the imaging of both granules and actin in super-resolution .
Our research centrally incorporates STED into a toolbox of multiple high resolution imaging techniques that we use, including total internal reflection microscopy, spinning disk and laser scanning confocal microscopy, and platinum replica electron microscopy. In addition, we have the ability to manipulate human NK cells by introducing fluorescent proteins and silencing constructs, allowing us to evaluate the function and regulation of critical NK cell proteins. Finally, we have access to patients with rare NK cell immunodeficiencies, giving us the unique perspective of human “knockouts”.
Challenges as we move forward include identification of fluorophores suitable for multiple channel STED and the development and optimization of fluorescent proteins durable enough to withstand depletion laser scanning (required for STED), which would allow for live cell super resolution imaging. As always, new technologies also result in new needs for data analysis and management, and we continue to develop quantitative algorithms for analysis of super-resolution images.
Recent advances allowing us to overcome the diffraction barrier have resulted in an explosion of nanoscopic technologies. The ability to “see” within the cell with such unprecedented resolution gives us the opportunity to visualize structures and proteins with previously unimaginable clarity. With this ability, we can continue to elucidate the mechanism behind NK cell processes, allowing us to further understand their role in human health and disease.