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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
Published on: December 29, 2017
Single-cell fluorescence imaging using metal plasmon-coupled probe 2: single-molecule counting on lifetime image
Jian Zhang1, Yi Fu, Dong Liang
1Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201, USA.
Nano Letters
|March 18, 2008
Summary
Novel metal plasmon-coupled probes (PCPs) significantly enhance fluorescence imaging of cell surfaces. These brighter, shorter-lifetime probes enable clearer visualization and quantification of cellular components.
Area of Science:
- Nanotechnology
- Biophotonics
- Cellular Imaging
Background:
- Concanavalin A (conA) is a lectin used for cell surface labeling.
- Autofluorescence from cells can obscure imaging signals.
- Enhancing fluorophore brightness and stability is crucial for advanced imaging.
Purpose of the Study:
- To synthesize and characterize metal plasmon-coupled probes (PCPs) for enhanced cellular imaging.
- To evaluate the imaging performance of PCPs compared to free fluorophores.
- To demonstrate the utility of PCPs for quantifying and visualizing cell surface components.
Main Methods:
- Covalent conjugation of Alexa Fluor 647-labeled conA to 20 nm silver nanoparticles to create PCPs.
- Fluorescence imaging using scanning confocal microscopy, analyzing both intensity and lifetime.
- Labeling of T-lymphocytic (PM1) cells with PCPs and analysis of emission spots.
Main Results:
- PCPs exhibited a 30-fold increase in brightness compared to free conA.
- PCPs showed a dramatically shortened fluorescence lifetime.
- Clear separation of PCP emission spots from cell autofluorescence was achieved.
- 3D scanning allowed for counting and distribution analysis of bound fluorophores.
Conclusions:
- Metal-associated fluorophores (PCPs) serve as effective molecular imaging agents.
- PCPs offer superior signal-to-noise ratio for cell surface imaging.
- PCPs enable quantification and spatial distribution analysis of cell surface components.
