Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells

Sora Yang, Nils Klughammer, Anders Barth, Marvin E. Tanenbaum*, Cees Dekker*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

10 Downloads (Pure)


Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate. We present a thorough theoretical and experimental characterization of the optical properties of these nanowells over a wide range of ZMW diameters and overmilling depths, showing an excellent signal confinement and a 5-fold fluorescence enhancement of fluorescent molecules inside nanowells. ZMW nanowells facilitate live-cell imaging as cells form stable protrusions into the nanowells. Importantly, the nanowells greatly reduce the cytoplasmic background fluorescence, enabling the detection of individual membrane-bound fluorophores in the presence of high cytoplasmic expression levels, which could not be achieved with TIRF microscopy. Zero-mode waveguide nanowells thus provide great potential to study individual proteins in living cells.

Original languageEnglish
Pages (from-to)20179-20193
Number of pages15
JournalACS Nano
Issue number20
Publication statusPublished - 2023


  • fluorescence correlation spectroscopy
  • fluorescence enhancement
  • fluorescence microscopy
  • live-cell imaging
  • palladium
  • single-molecule fluorescence
  • zero-mode waveguide


Dive into the research topics of 'Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells'. Together they form a unique fingerprint.

Cite this