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Dual-Labeled Graphene Quantum Dot-Based Forster Resonance Energy Transfer Nanoprobes for Single-Molecule Localization Microscopy.

ABSTRACT: Single-molecule localization microscopy (SMLM)-based super-resolution imaging techniques (e.g., photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) require that the employed optical nanoprobes possess fluorescence intensity fluctuations under certain excitation conditions. Here, we present a dual-labeled graphene quantum dot (GQD)-based Förster resonance energy transfer (FRET) nanoprobe, which is suitable for SMLM imaging. The nanoprobe is constructed by attaching Alexa Fluor 488 (AF488) and Alexa Fluor 568 (AF568) dye molecules onto GQDs. Experimental results confirmed the FRET effect of the nanoprobes. Moreover, under a single 405 nm excitation, the FRET nanoprobe exhibits excellent blinking behavior. SMLM imaging of microtubules in MRC-5 cells is realized. The presented nanoprobe shows great potential in multicolor SMLM-based super-resolution imaging.

SUBMITTER: Lu J

PROVIDER: S-EPMC8028002 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Dual-Labeled Graphene Quantum Dot-Based Förster Resonance Energy Transfer Nanoprobes for Single-Molecule Localization Microscopy.

Lu Ju J Zong Shenfei S Wang Zhuyuan Z Chen Chen C Zhang Yizhi Y Wang Hong H Cui Yiping Y

ACS omega 20210324 13

Single-molecule localization microscopy (SMLM)-based super-resolution imaging techniques (<i>e.g.</i>, photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM)) require that the employed optical nanoprobes possess fluorescence intensity fluctuations under certain excitation conditions. Here, we present a dual-labeled graphene quantum dot (GQD)-based Förster resonance energy transfer (FRET) nanoprobe, which is suitable for SMLM imaging. The nanoprobe is c ...[more]

PMID: 33842752

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Project description:BackgroundAntimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial.ResultsHere we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level.ConclusionRecently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process: 1) Binding, mediated mainly by charged residues in the peptide; 2) Peptide association, as peptide concentration increases evidenced by a change in diffusive behavior; 3) Membrane disruption, during which lipopolysaccharide is not released; and 4) Lysis, by leakage of cytosolic content through large membrane defects.

Dataset Information

Dual-Labeled Graphene Quantum Dot-Based Forster Resonance Energy Transfer Nanoprobes for Single-Molecule Localization Microscopy.

Publications

Dual-Labeled Graphene Quantum Dot-Based Förster Resonance Energy Transfer Nanoprobes for Single-Molecule Localization Microscopy.

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