한빛사
>
한빛사논문
한빛사논문
- 조회116
ACS Nano, Oct 24 2023, 17 (20) | https://doi.org/10.1021/acsnano.3c05959
Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells
Sora Yang 1, Nils Klughammer 2, Anders Barth 2, Marvin E Tanenbaum 1 2 *, Cees Dekker 2 *
1Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
2Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
2Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
*Corresponding authors: correspondence to Marvin E Tanenbaum or Cees Dekker
Abstract
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.
논문정보
- Research article
- 2023년 10월 (BRIC 등록일 2025-02-05)
- 국외 연구진
댓글 작성 로그인
팝업 닫기관련 링크
