Abstract
Abstract Analysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.
Highlights
Exploration of the single-molecule processes of the nucleus in living cells is important for understanding complex biological systems [1,2,3,4]
To confirm the physical interaction between the surface of hybrid nanopillar arrays (HNPs) and cells, Scanning electron microscopy (SEM) images were taken to verify the morphological change in the cell shape when cultured on HNPs and the structural changes of the individual HNPs in close physical contact with the cells
We have shown that the vertical HNP structures meet the requirements for use as a probe to detect the localized fluorescence signal at the Au caps of HNPs at the interface of cells-HNP substrates
Summary
Exploration of the single-molecule processes of the nucleus in living cells is important for understanding complex biological systems [1,2,3,4]. Fluorescence-based technologies have been used to study the structural and dynamic properties of biological proteins, such as ion channels, receptors, and transporters [5,6,7,8]. They have become a powerful tool for understanding protein–protein interactions that represent specific information about the time trajectory of proteins that interact with other related proteins and the basic steps of the reaction mechanisms. The nanopillars affect locally curving of plasma membranes and upward bending of nuclear membrane of the cells adherent to the
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