دورية أكاديمية
أعرض في EDS

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

التفاصيل البيبلوغرافية
العنوان: Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.
المؤلفون: Yang S; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands., Klughammer N; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands., Barth A; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands., Tanenbaum ME; Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands., Dekker C; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
المصدر: ACS nano [ACS Nano] 2023 Oct 24; Vol. 17 (20), pp. 20179-20193. Date of Electronic Publication: 2023 Oct 04.
نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 101313589 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1936-086X (Electronic) Linking ISSN: 19360851 NLM ISO Abbreviation: ACS Nano Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington D.C. : American Chemical Society
مواضيع طبية MeSH: Nanotechnology*/methods , Microscopy*, Single Molecule Imaging ; Spectrometry, Fluorescence/methods
مستخلص: 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.
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فهرسة مساهمة: Keywords: fluorescence correlation spectroscopy; fluorescence enhancement; fluorescence microscopy; live-cell imaging; palladium; single-molecule fluorescence; zero-mode waveguide
تواريخ الأحداث: Date Created: 20231004 Date Completed: 20231026 Latest Revision: 20231029
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC10604100
DOI: 10.1021/acsnano.3c05959
PMID: 37791900
قاعدة البيانات: MEDLINE
الوصف
تدمد:1936-086X
DOI:10.1021/acsnano.3c05959