Nanophotonic approaches for nanoscale imaging and single‐molecule detection at ultrahigh concentrations

Abstract

Over the last decade, we have witnessed an outburst of many different optical techniques aimed at breaking the diffraction limit of light, providing super-resolution imaging on intact fixed cells. In parallel, single-molecule detection by means of fluorescence has become a common tool to investigate biological interactions at the molecular level both in vitro and in living cells. Despite these advances, visualization of dynamic events at relevant physiological concentrations at the nanometer scale remains challenging. In this review, we focus on recent advancements in the field of nanophotonics toward nanoimaging and single-molecule detection at ultrahigh sample concentrations. These approaches rely on the use of metal nanostructures known as optical antennas to localize and manipulate optical fields at the nanometer scale. We highlight examples on how different optical antenna geometries are being implemented for nanoscale imaging of cell membrane components. We also discuss different implementations of selfstanding and two-dimensional antenna arrays for studying nanoscale dynamics in living cell membranes as well as detection of individual biomolecular interactions in the mM range for sensing applications. Microsc.

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

“…Practically speaking, this barrier can be overcome by for example reducing the effective observation volume e.g., using lipid vesicles. Alternatively, nanophotonic structures have been utilized to either further confine the observation volume (e.g., zero-mode waveguides) or to enhance the fluorescence of a single-molecule attached in a measurement hot-spot (reviewed for example here [149,150]). Zero-mode waveguides are currently utilized for single-molecule sequencing instruments [151].…”

Large-Scale Arrays of Bowtie Nanoaperture Antennas for Nanoscale Dynamics in Living Cell Membranes

“…Single-molecule detection has become a powerful tool to study biological processes both in vitro and in living cells at diluted sample concentrations. − However, traditional single molecule methods rely on diffraction-limited optics, which pose a challenge to the study of individual dynamic events at relevant physiological concentrations . Advances in the field of nanophotonics are starting to provide elegant strategies to reduce the excitation volume beyond diffraction allowing in vitro detection of single molecules at nearly physiological concentrations (μM range). , For instance, zero-mode-waveguides (ZMW) consisting of nanoapertures (∼150 nm in size) on metallic substrates , have been used in a large number of single molecule in vitro assays − and in some live cell experiments. , More recently, single molecule analysis in solution at 20 μM sample concentrations has been impressively demonstrated using plasmonic optical antennas, which synergistically combine large enhancement and confinement of the light down to nanoscale volume . The application of these devices to live cell research would indisputably open up new possibilities to investigate in real time multimolecular processes at physiological expression levels.…”

“…4 Advances in the field of nanophotonics are starting to provide elegant strategies to reduce the excitation volume beyond diffraction allowing in vitro detection of single molecules at nearly physiological concentrations (μM range). 4,5 For instance, zero-mode-waveguides (ZMW) consisting of nanoapertures (∼150 nm in size) on metallic substrates 6,7 have been used in a large number of single molecule in vitro assays 6−11 and in some live cell experiments. 12,13 More recently, single molecule analysis in solution at 20 μM sample concentrations has been impressively demonstrated using plasmonic optical antennas, which synergistically combine large enhancement and confinement of the light down to nanoscale volume.…”

Roadmap on biosensing and photonics with advanced nano-optical methods

“…Nanophotonic approaches have recently emerged as attractive tools to investigate the nanoscale dynamics of living cell membranes, potentially overcoming most of the barriers associated with diffraction-limited and super-resolution methods. In particular, metallic nanostructures known as photonic antennas hold great potential for biology as they both confine and amplify optical fields at the nanometer scale (figure 12) [50,51]. Moreover, they can be designed for broadband operation allowing for multi-color light confinement in the visible regime [17].…”