Variable-Angle Nanoplasmonic Fluorescence Microscopy: An Axially Resolved Method for Tracking the Endocytic Pathway

Abstract

The study of endocytosis, which encompasses diverse mechanisms in biology, requires the utilization of high axial resolution to monitor molecular behavior on both the cell surface and interior of the cell. We have designed a novel axially resolved fluorescence microscopic technique, termed variable-angle nanoplasmonic fluorescence microscopy. The proof-of-principle of this approach is achieved by selectively following the events in the vicinity of a cell membrane or in a cell. We use a 30 nm Au-coated semitransparent coverslip as the nanoplasmonic chip to achieve both surface plasmon resonance excitation and critical angle excitation by tuning the incident angles. This approach leads to improved axial resolution compared to total internal reflection fluorescence microscopy, which is a common imaging technique in cell biology. It offers a unique opportunity to semiquantitatively determine fluorophore axial distributions in the cell. Observing the epidermal growth factor receptor-mediated endocytosis in Caski cells clearly demonstrates the potential application of this new method for cell biology studies.

Surface plasmon–coupled emission imaging for biological applications

“…Recently, our group developed a novel imaging approach, termed as variable-angle nanoplasmonic fluorescence microscopy (VANFM), to selectively modulate the imaging depth with an improved axial resolution [81]. As SPCEM provides better axial confinement in the extremely close vicinity, we expect a higher imaging resolution in the axial direction with VANFM.…”

“…Previously, our group has provided a study to explore the cross-scale effect of SPCE and demonstrated its ability to study macro-scale cells [87]. Afterwards, we proposed the axial-resolved VANFM to release the axial restriction and applied it to observing the cellular activity [81].…”

“…10 Angle-dependent fluorescence intensity, fluorescence images, and schematics of the ligand-receptor complex distributions after recovery at room temperature for 0 min and 30 min, respectively (scale bars, 20 μm) (reprinted with permission from ref. [81]. Copyright © (2019) American Chemical Society)…”

Amplified Fluorescence by Hollow-Porous Plasmonic Assembly: A New Observation and Its Application in Multiwavelength Simultaneous Detection

“…As important spectral technologies, fluorescence-based techniques have been widely used in all kinds of analysis and detection, but insufficient sensitivity limits their further development. − It has been shown that the radiation decay rate and the distribution of radiation energy of fluorophores can be changed when they are placed on a metal surface or a metal surface with nanostructures, which enables the increase of sensitivity. A new technique, namely, surface plasmon coupled emission (SPCE), in which directional and enhanced radiation is produced by the interaction between an excited fluorophore in the near-field (20–200 nm) of a continuous metallic surface and surface plasmons (SPs), − has been developed and demonstrated to be useful in biochemical detection − and imaging , due to its distance dependence and wavelength resolution.…”

Plasmon Coupling Enhanced Micro‐Spectroscopy and Imaging for Sensitive Discrimination of Membrane Domains of a Single Cell

“…[34][35][36] Researchers, including our group, have utilized SPCE to study basal cellular membrane-related activities. [37][38][39][40][41] However, previous studies focused on investigating a single membrane domain, which is of no help for simultaneously determining the role of membrane domains in cellular functions. Thus, it is highly desirable to develop a microscopic technique capable of identifying and characterizing the basal and lateral membrane domains.…”