Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution

Super-resolution fluorescence imaging that surpasses the classical optical resolution limit is widely utilized for resolving the spatial organization of biological structures at molecular length scales. In one example, single-molecule localization microscopy, the lateral positions of single molecules can be determined more precisely than the diffraction limit if the camera collects individual photons separately. Using several schemes that introduce engineered optical aberrations in the imaging optics, super-resolution along the optical axis (perpendicular to the sample plane) has been achieved, and single-molecule localization microscopy has been successfully applied for the study of 3D biological structures. Nonetheless, the achievable axial localization accuracy is typically three to five times worse than the lateral localization accuracy. Only a few exceptional methods based on interferometry exist that reach nanometer 3D super-resolution, but they involve enormous technical complexity and restricted sample preparations that inhibit their widespread application. We developed metal-induced energy transfer imaging for localizing fluorophores along the axial direction with nanometer accuracy, using only a conventional fluorescence lifetime imaging microscope. In metal-induced energy transfer, experimentally measured fluorescence lifetime values increase linearly with axial distance in the range of 0-100 nm, making it possible to calculate their axial position using a theoretical model. If graphene is used instead of the metal (graphene-induced energy transfer), the same range of lifetime values occurs over a shorter axial distance (similar to 25 nm), meaning that it is possible to get very accurate axial information at the scale of a membrane bilayer or a molecular complex in a membrane. Here, we provide a step-by-step protocol for metal- and graphene-induced energy transfer imaging in single molecules, supported lipid bilayer and live-cell membranes. Depending on the sample preparation time, the complete duration of the protocol is 1-3 d.

Automated summary

Super-resolution fluorescence imaging is commonly used to study the spatial organization of biological structures at molecular scales, achieving resolutions beyond the classical optical limit. Various methods have been developed for super-resolution imaging along the optical axis, with some reaching nanometer accuracy in 3D localization. However, these approaches can be technically complex and have limited applicability. Metal-induced energy transfer imaging offers a simpler alternative for nanometer-scale axial localization of fluorophores.