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Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy
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Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.

Sora Yang1, Nils Klughammer2, Anders Barth2

  • 1Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.

ACS Nano
|October 4, 2023
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Summary

Researchers developed overmilled zero-mode waveguides (ZMWs) to enable single-molecule imaging in live cells. These nanowells reduce background noise, allowing visualization of individual proteins even with high intracellular concentrations.

Keywords:
fluorescence correlation spectroscopyfluorescence enhancementfluorescence microscopylive-cell imagingpalladiumsingle-molecule fluorescencezero-mode waveguide

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Cell Biology

Background:

  • Single-molecule fluorescence imaging typically requires very low protein concentrations (sub-nanomolar).
  • High intracellular protein concentrations in live cells pose a significant challenge for isolating single molecules.
  • Existing techniques like Total Internal Reflection Fluorescence (TIRF) microscopy struggle with high background fluorescence.

Purpose of the Study:

  • To develop a method for achieving single-molecule observations within live cells.
  • To overcome the limitations of high intracellular protein concentrations in live-cell imaging.
  • To enable the detection of individual proteins, particularly membrane-bound ones, in a high-background cellular environment.

Main Methods:

  • Utilized overmilled zero-mode waveguides (ZMWs) to create tunable nanowells.
  • Characterized the optical properties of ZMW nanowells theoretically and experimentally.
  • Investigated cell penetration and protrusion formation into the ZMW nanowells.
  • Compared ZMW nanowell performance against Total Internal Reflection Fluorescence (TIRF) microscopy.

Main Results:

  • Overmilling ZMWs in a palladium film created tunable nanowells allowing cell penetration.
  • Demonstrated excellent signal confinement and a 5-fold fluorescence enhancement within nanowells.
  • Observed stable cell protrusions into the ZMW nanowells, facilitating live-cell imaging.
  • Successfully reduced cytoplasmic background fluorescence, enabling detection of single membrane-bound fluorophores.

Conclusions:

  • Overmilled ZMW nanowells significantly reduce observation volume and background noise.
  • This technique enables single-molecule imaging in live cells, overcoming high cytoplasmic concentrations.
  • ZMW nanowells offer a promising approach for studying individual proteins in their native cellular environment.