MAxSIM: Multi-angle-crossing structured illumination microscopy with height-controlled mirror for 3D topological mapping of live cells.

Abstract

The plasma membrane sheet can fold into dynamic, three-dimensional (3D) structures that correlate with cell functionality. Mapping 3D plasma membrane topology in live cells can provide unprecedented insights into cell biology and signaling. Fluorescence light interference contrast (FLIC) microscopy and its derivatives (e.g., scanning angle interference microscopy) use axial interferometry to determine nanoscale axial locations of cell-surface proteins that are placed on a mirror along the optical axis. However, FLIC derivatives have limitations including low fidelity/poor axial localization and low time resolution due to slow mechanical control of incident angles, cell placement on a mirror, and an unoptimized data fitting algorithm, as well as diffraction-limited lateral imaging. To resolve these challenges, we developed multi-angle-crossing structured illumination microscopy (MAxSIM). MAxSIM generates multiple incident angles by optoelectronically creating diffraction patterns as in structured illumination microscopy to enable super-resolution lateral imaging. MAxSIM performance is enhanced with a custom-fabricated height-controlled mirror (HCM) placed at a fixed location from the bottom glass substrate, which significantly increases axial localization fidelity and optimizes the height reconstruction scheme. Use of HCM additionally enables cell placement on the glass side of the bottom substrate and flexible cell imaging from both the top and bottom of a cell. Our MAxSIM platform with an HCM and optimized reconstruction scheme thus offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes and enables near-real-time (∼1 Hz) imaging of live cells and (∼20 Hz) 3D single molecule tracking. These innovations will significantly advance basic biological research by providing new and widely applicable tools that enable novel 3D measurements of cell-surface topologies that will reveal unprecedented mechanistic details of the control of cellular functions.