Axial Scanning Metal‐Induced Energy Transfer Microscopy for Extended Range Nanometer‐Sectioning Cell Imaging

Abstract

Nanometer‐sectioning optical microscopy has become an indispensable tool in membrane‐related biomedical studies. Finally, many nanometer‐sectioning imaging schemes, such as variable‐angle total internal reflection fluorescence microscopy, metal‐induced energy transfer (MIET) imaging, and supercritical‐angle fluorescence microscopy have been introduced. However, these methods can measure a single layer of molecules, and the measurement ranges are below 100 nm, which is not large enough to cover the thickness of lamellipodium. This paper proposes an optical imaging scheme that can identify the axial locations of two layers of molecules with an extended measurement range and a nanometer‐scale precision by using MIET, axial focal plane scanning, and biexponential analysis in fluorescence lifetime imaging microscopy. The feasibility of the proposed method is demonstrated by measuring an artificial sample of a known structure and the lamellipodium of a human aortic endothelial cell whose thickness ranges from 100 to 450 nm with 18.3 nm precision.

Measuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy

“…In previous studies, MIET has been primarily used for structural analysis, such as mapping the topography of the basal membrane of living cells 31 , reconstructing focal adhesions and stress fibers in three dimensions 34 , measuring the distance between the inner and outer envelope of the nucleus 35 , visualizing the dynamics of epithelial-mesenchymal transitions (EMT) 36 , and enabling single-molecule localization and co-localization along the optical axis 37 , 38 . Additionally, MIET has been utilized to map the basal membrane and lamellipodia of human aortic endothelial cells 39 , 40 and to achieve three-dimensional isotropic resolution imaging of microtubules and clathrin pits in combination with SMLM 41 . By replacing the thin metal film with a single sheet of graphene (Graphene-Induced Energy Transfer or GIET), the localization accuracy can be improved by an order of magnitude within ~25 nm from the substrate 32 .…”

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins

“…The localization accuracy depends on the number of detected photons which determines the accuracy of the determined fluorescence lifetime, and with a few thousand of detected photons one typically achieves an axial resolution down to a few nanometers. Previous applications of MIET imaging include mapping the topography of the basal membrane of living cells, 24 threedimensional reconstruction of focal adhesions and stress fibers, 26 measuring the distance between the inner and outer envelope of the nucleus, 27 visualizing the dynamics of epithelial−mesenchymal transitions (EMT), 28 single-molecule localization and colocalization, 29,30 mapping the basal membrane and lamellipodia of human aortic endothelial cells, 31,32 and, in combination with SMLM, three-dimensional isotropic resolution imaging of microtubules and clathrin pits. 33 Recently, it was shown that substituting the metal layer with a single sheet of graphene (graphene-induced energy transfer or GIET imaging) achieves a ca.…”

Measuring sub-nanometer fluctuations at microsecond temporal resolution with metal-and graphene-induced energy transfer spectroscopy

“…In previous studies, MIET has been primarily used for structural analysis, such as mapping the topography of the basal membrane of living cells [26], reconstructing focal adhesions and stress fibers in three dimensions [29], measuring the distance between the inner and outer envelope of the nucleus [30], visualizing the dynamics of epithelial-mesenchymal transitions (EMT) [31], and enabling single-molecule localization and co-localization along the optical axis [32,33]. Additionally, MIET has been utilized to map the basal membrane and lamellipodia of human aortic endothelial cells [34,35] and to achieve three-dimensional isotropic resolution imaging of microtubules and clathrin pits in combination with SMLM [36]. By replacing the thin metal film with a single sheet of graphene (Graphene-Induced Energy Transfer or GIET), the localization accuracy can be improved by an order of magnitude within ∼25 nm from the substrate [27].…”