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 (~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.

超越经典光学分辨率极限的超分辨率荧光成像被广泛用于解析分子长度尺度的生物结构的空间组织。在单分子定位显微镜的一个例子中，如果相机分别收集单个光子，则可以比衍射极限更精确地确定单个分子的横向位置。使用在成像光学中引入工程光学像差的几种方案，实现了沿光轴（垂直于样品平面）的超分辨率，并且单分子定位显微镜已成功应用于 3D 生物结构的研究。尽管如此，可实现的轴向定位精度通常比横向定位精度差三到五倍。只有少数基于干涉测量的特殊方法可以达到纳米 3D 超分辨率，但它们涉及巨大的技术复杂性和受限的样品制备，阻碍了它们的广泛应用。我们仅使用传统的荧光寿命成像显微镜开发了金属诱导能量转移成像，用于以纳米精度沿轴向定位荧光团。在金属诱导的能量转移中，实验测量的荧光寿命值在 0-100 nm 范围内随轴向距离线性增加，从而可以使用理论模型计算它们的轴向位置。如果使用石墨烯代替金属（石墨烯诱导的能量转移），则在较短的轴向距离（~25 nm）内会出现相同的寿命值范围，这意味着可以在膜双层或膜中的分子复合物的尺度上获得非常准确的轴向信息。在这里，我们为单分子、支持的脂质双层和活细胞膜中的金属和石墨烯诱导的能量转移成像提供了一个分步协议。根据样品制备时间，协议的完整持续时间为 1-3 天。