Localization-Based Super-Resolution Imaging: Methods and Biological Applications
Super-resolution microscopy has revolutionized the use of fluorescence imaging for biological applications. Localization-based methods such as fluorescence photoactivation localization microscopy (FPALM) stochastically activate and image sparse subsets of fluorescent molecules within a specimen, and then use the acquired images to determine the position of each molecule with nanometer precision. Many sparse subsets of molecules are imaged successively, and the coordinates of many molecules obtained over time are then plotted together as an image. These methods provide a means of imaging biological specimens, including living cells, with resolution in the tens of nanometers and time resolution of less than one second per rendered image. Methodologies to image samples with multiple labels, to image in three dimensions, and to image molecular orientations, have also been developed. These powerful capabilities have enabled a large number of important biological questions to potentially be addressed.
In addition to developing the methods themselves, we have applied FPALM to study the organization of biological membranes, and in particular the interactions between the influenza viral membrane protein hemagglutinin (HA) and the host cell actin cytoskeleton. HA is required at high concentrations on virion and host cell membranes for infectivity. As the role of actin in membrane organization is incompletely understood, we quantified the relationship between HA and host cell actin at the nanoscale, with the goal of identifying new approaches to block infection. Results using FPALM in non-polarized cells show HA clusters colocalize with actin-rich membrane regions (ARMR). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMR, and actin disruption caused significant changes in HA clustering. The actin binding protein cofilin was sometimes excluded from regions within several hundred nanometers of HA clusters, suggesting that HA or adjacent proteins in the same clusters influence local actin structure.
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