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

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

Research output: Contribution to journal/periodicalArticleScientificpeer-review


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 - 24 Oct 2023


  • Nanotechnology/methods
  • Microscopy
  • Single Molecule Imaging
  • Spectrometry, Fluorescence/methods


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