Abstract
AbstractMapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking.
Funder
National Science Foundation
GW | Dr. Cyrus and Myrtle Katzen Cancer Research Center, George Washington University
GW Technology Maturation Grant
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
Reference57 articles.
1. Edidin, M. The state of lipid rafts: From model membranes to cells. Annu Rev. Bioph Biom. 32, 257–283 (2003).
2. van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Bio 9, 112–124 (2008).
3. Wu, Y. M. et al. Contacts between the endoplasmic reticulum and other membranes in neurons. Proc. Natl Acad. Sci. USA 114, E4859–E4867 (2017).
4. Graham, T. R. & Kozlov, M. M. Interplay of proteins and lipids in generating membrane curvature. Curr. Opin. Cell Biol. 22, 430–436 (2010).
5. de la Serna, J. B., Schutz, G. J., Eggeling, C. & Cebecauer, M. There is no simple model of the plasma membrane organization. Front. Cell Dev. Biol. 4, 106 (2016).