Dynamic, nanoscale interactions on and near the plasma membrane of live cells participate in critical cell biological functions (e.g. cell adhesion and migration), but many aspects are not yet described due to the state of investigative tools. Applicable tools like Scanning Angle Interference Microscopy (SAIM) can perform nanoscale 3D localization in the plasma membrane. However, the current version of SAIM only works on fixed or ruptured cells and does not allow for real-time monitoring of topological dynamics in live cells. Moreover, its applicability has been limited to the edge of the apical cell regions.
GW researchers increased measurement accuracy, enabled near real-time live-cell measurements, and enlarged the measurable area for axial localization with a novel height-controlled mirror (sample holder). Previously, the sample geometry required cells laid on a top mirror with an undefined distance between the bottom coverglass and the top mirror, which limits the imaging areas to be at the cell edge and disables real-time monitoring of cell topology. These limitations were resolved by the new sample holder wherein the distance between the mirror and coverglass is defined by ridges of a specific height. Moreover, the new sample holder enables versatile cell placement (either at the top mirror or the bottom coverglass) and support for electron microscopy validation studies. The production is currently at lab-scale.
A schematic of the SAIM system with the novel height-controlled mirror.
• Monitor near-real-time (~1 Hz) 3D changes of nanometer-scale membrane topology and protein distribution in both live and fixed cells • Visualize proteins on intracellular organelles near cell surface with 3D nanoscale topology
• Higher detection rate (near real-time) and higher axial localization accuracy than conventional SAIM • Enables live-cell imaging • Accurate axial localization for thicker portions of a cell than previously possible • Sample placement versatility • Ready for confirmation by electron microscopy
MAxSIM: multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells. Pedro Felipe Gardeazabal Rodriguez, Yigal Lilach, Abhijit Ambegaonkar, Teresa Vitali, Haani Jafri, Hae Won Sohn, Matthew Dalva, Susan Pierce & Inhee Chung. Communications Biology volume 6, Article number: 1034 (2023).