Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >106 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show that this localized diffusion slowdown is not due to altered lipid packing order but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.
Keyphrases
- single molecule
- atomic force microscopy
- living cells
- endoplasmic reticulum
- high resolution
- estrogen receptor
- escherichia coli
- breast cancer cells
- high density
- high speed
- fatty acid
- locally advanced
- squamous cell carcinoma
- brain injury
- fluorescence imaging
- sensitive detection
- binding protein
- psychometric properties
- cerebral ischemia
- subarachnoid hemorrhage
- single cell