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

TY - JOUR

T1 - Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells

AU - Yang, Sora

AU - Klughammer, Nils

AU - Barth, Anders

AU - Tanenbaum, Marvin E

AU - Dekker, Cees

PY - 2023/10/24

Y1 - 2023/10/24

N2 - 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.

AB - 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.

KW - Nanotechnology/methods

KW - Microscopy

KW - Single Molecule Imaging

KW - Spectrometry, Fluorescence/methods

U2 - 10.1021/acsnano.3c05959

DO - 10.1021/acsnano.3c05959

M3 - Article

C2 - 37791900

SN - 1936-0851

VL - 17

SP - 20179

EP - 20193

JO - ACS nano

JF - ACS nano

IS - 20

ER -