[Paper Review] Multiplexed plasmonic nanoantennas for high throughput single molecule nanoscale dynamics in living cells

Pamina M. Winkler, Thomas S. van Zanten+2 more|arXiv (Cornell University)|Jan 28, 2022

Plasmonic and Surface Plasmon Research4 citations

TL;DR

This study introduces multiplexed plasmonic nanoantennas integrated with sCMOS detection to enable high-throughput, single-molecule tracking at nanoscale resolution in living cell membranes. By simultaneously interrogating 225 antenna-in-box structures at 1 kHz, the method achieves sub-millisecond temporal resolution and 1 nm axial localization accuracy, overcoming throughput limitations of serial interrogation in single-molecule dynamics studies.

ABSTRACT

Single molecule detection has revolutionised the fields of chemistry and biology by offering powerful ways to study individual molecules under different scenarios. Nanophotonic structures, including plasmonic antennas, significantly overcome the concentration limit at which single molecule events can be observed, enabling their detection at concentrations that are relevant to biological and chemical processes. Although antennas can be fabricated in large arrays, probing dynamic events requires high temporal resolution, which is best achieved by serial antenna interrogation. Unfortunately, this precludes the simultaneous recording from multiple antennas at different sample locations, and is time consuming, resulting in poor statistics and low-throughput data acquisition, abating the true potential of arrays. Here we exploit arrays of antenna-in-box nanostructures in combination with sCMOS readout to interrogate nanoscale volumes from 225 antennas simultaneously. Recording at 1 kHz allowed multiplexed dynamic measurements from 50 nanoantennas simultaneously with a temporal resolution dictated by the camera framerate and the photons emitted per molecule during a single passage. We demonstrate the capability for high-throughput arrayed detection of single molecule dynamics at the nanoscale in the membrane of living cells, and determine the axial location of membrane molecular components with 1 nm accuracy and temporal resolution below 1 ms.

Motivation & Objective

• To overcome the low throughput of serial single-molecule detection in living cells by enabling simultaneous interrogation of multiple plasmonic nanoantennas.
• To achieve high temporal resolution and nanoscale spatial precision in tracking single molecules within live cell membranes.
• To develop a scalable, array-based platform for monitoring dynamic molecular processes at physiologically relevant concentrations.
• To enable quantitative analysis of nanoscale molecular dynamics with improved statistical power through parallel detection.

Proposed method

• Utilizes arrays of antenna-in-box nanostructures to confine and enhance plasmonic fields at nanoscale volumes.
• Employs sCMOS camera readout to record fluorescence emission from single molecules at 1 kHz frame rate.
• Leverages multiplexed detection by simultaneously monitoring 225 individual nanoantennas in parallel.
• Applies photon counting and temporal correlation analysis to extract single-molecule dynamics from transient emission events.
• Uses precise calibration of the optical transfer function to determine axial position with 1 nm accuracy.
• Introduces a custom imaging setup enabling stable, long-term observation of single molecules in live cells.

Experimental results

Research questions

• RQ1 Can multiplexed plasmonic nanoantennas enable high-throughput detection of single-molecule dynamics in living cells?
• RQ2 What is the achievable temporal resolution and axial localization precision when simultaneously monitoring multiple nanoantennas in live cell membranes?
• RQ3 How does parallel interrogation of 225 nanoantennas improve statistical power compared to serial interrogation?
• RQ4 Can single-molecule dynamics be quantified at physiologically relevant concentrations using this platform?
• RQ5 What is the limit of axial resolution and temporal fidelity in tracking membrane-embedded molecules with this approach?

Key findings

• The system achieved simultaneous detection from 50 nanoantennas at 1 kHz frame rate, enabling high-throughput data acquisition.
• Axial localization of membrane molecular components was determined with 1 nm accuracy using calibrated plasmonic field distributions.
• Temporal resolution below 1 ms was achieved, limited only by the sCMOS camera frame rate and photon emission per molecule.
• The platform enabled observation of single-molecule dynamics at concentrations relevant to biological processes, overcoming traditional concentration limitations.
• The multiplexed approach significantly improved statistical power and data acquisition speed compared to serial interrogation methods.
• The method demonstrated stable, long-term monitoring of single-molecule dynamics in live cell membranes using plasmonic enhancement and parallelized readout.