Near-Field Imaging of Cell Membranes in Liquid Enabled by Active Scanning Probe Mechanical Resonance Control

Abstract

Despite the power of far-field super-resolution microscopies for three-dimensional imaging of biomolecular structures and processes, its application is challenged in dense and crowded samples and for certain surface and membrane studies. Although near-field imaging with its ability to provide intrinsic subdiffraction limited spatial resolution at any optical modality, its application to biological systems has remained limited because of the difficulties of routine operation in liquid environments. Here we demonstrate stable and sensitive near-field scanning optical microscopy (NSOM) in a liquid based on a new mechanical resonance control and an optimization of the tip length, achieving a high quality factor (>2800) force sensing of the near-field probe. Through near-field imaging of the spatial distribution of epidermal growth factor receptors (EGFRs) on the membrane of A431 cancer cells as an example, we reveal nanoscale correlations between surface EGFR and intracellular organelle structures with ∼50 nm spatial resolution. The method provides a new avenue for surface imaging in viscous liquid media to complement super-resolution microscopy for studies of biological membranes, nanostructures, and interfaces.

Tip-enhanced photoluminescence nano-spectroscopy and nano-imaging

“…This approach enabled to resolve individual Ca 2+ pumps and PMCA4 proteins on the cell membrane in PBS solution with 50 nm spatial resolutions [108,117]. The inverse approach of near-field biological imaging in liquid was also demonstrated [33]. This study showed a conventional NSOM probe can be operated in liquid by increasing Qfactor with a novel resonance control method.…”

“…The metal-coated optical fiber probe has a nano-aperture with a diameter of ∼50-100 nm, which determines a spatial resolution of NSOM, to detect or to illuminate near-field signals on the surface of samples [27]. With a high spatial resolution far beyond the diffraction limit, many groundbreaking works were allowed in physics [28,29], chemistry [30,31], biology [32,33], and material sciences [34][35][36][37]. However, against all expectations, the application range of NSOM had been highly limited due to the low sensitivity, since the transmission intensity through the NSOM probe is inversely proportional to the fourth power of the aperture size [38,39].…”

Visualization and measurement of the local absorption coefficients of single bilayer phospholipid membranes using scanning near-field optical microscopy

“…To further enhance the potential applications of SNOM for more complex biological systems, simultaneous spectroscopic measurements can be carried out which will provide additional information about the samples' chemical structures. In addition, water-based SNOM scanning is also possible with careful characterization of the control mechanism in the probe oscillation for more biocompatible samples [51,52].…”

Graphene Coated Nanoprobes: A Review

“…To do so, the metallic substrate (typically a foil) is introduced in a tube CVD furnace and heated at high temperatures (>850 • C) while the graphene precursor (typically CH 4 or C 2 H 5 OH) is introduced in the tube with the assistance of a carrier gas (typically H 2 /Ar). Using this approach, the precursor seeds (carbon-containing molecules) can precipitate at random locations on the surface of the metallic sample, and they grow until merging into each other, forming a homogeneous (but polycrystalline) graphene film-intuitively this process is similar to placing several ice cubes on a flat table and waiting until they melt and form a homogeneous water film.…”

“…Sharp probe tips with an apex radius <100 nm are widely used in many different fields of science including electronics [1], physics [2], chemistry [3], biology [4] and medicine [5], as they allow local characterization and manipulation of materials with a nanometric spatial resolution. Depending on the equipment and application in which they are used, nanoprobes can have a wide variety of shapes, material composition and prizes.…”