AI Article Synopsis
- Individual Cy5 fluorophores attached to DNA were studied using single-molecule spectroscopy to measure their blinking behavior under deoxygenated conditions, revealing spontaneous on/off fluctuations lasting seconds.
- The blinking of Cy5 is influenced by factors such as the donor's proximity, structure, and the presence of dual wavelength excitation light (514 nm and 640 nm).
- By utilizing an alternating laser excitation strategy, researchers can effectively distinguish between the dark states of Cy5 blinking and fluctuations due to FRET, addressing issues with slow blinking that coincide with critical biological time scales.
Article Abstract
The blinking kinetics of individual Cy5 fluorophores conjugated to DNA are directly measured using single-molecule spectroscopy. Under deoxygenated aqueous conditions, Cy5 fluorescence exhibits spontaneous and reversible on/off fluctuations with a period lasting seconds. This blinking is observed when directly exciting Cy5 with 640 nm light and by Forster resonance energy transfer (FRET). We find that Cy5 blinking is influenced by the proximity of the donor, the structure of the donor, the presence of 514 nm excitation, and FRET. In the context of single-molecule FRET, blinking of the acceptor produces anticorrelated donor-acceptor intensity fluctuations, which can be difficult to discern from variations in the interdye distance. Slow blinking is, in particular, problematic because it overlaps with biologically relevant time scales. By employing an alternating 514640 nm laser excitation scheme, we show that the dark states can be readily resolved and discriminated from FRET distance fluctuations.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1063/1.2136157 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Acceptance and willingness of patients with chronic facial nerve palsy for an implantable device that assists with eye closure.
J Plast Reconstr Aesthet Surg
September 2024
Department of Head and Neck Surgery, Chris O'Brien Lifehouse, Camperdown, Sydney, NSW, Australia; Sydney Medical School, Faculty of Medicine and Health, University of Sydney, Camperdown, Sydney, NSW, Australia; Sydney Facial Nerve Clinic, The Chris O'Brien Lifehouse, Camperdown, NSW 2050, Australia; Department of Otolaryngology-Head & Neck Surgery, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Background: Patients with facial nerve palsy often experience lagophthalmos (incomplete eye closure), which can lead to exposure keratitis. The Bionic Lid Implant for Natural Eye Closure (BLINC) is a medical device designed to mimic the more natural blink kinetics than traditional lid loading techniques.
Aims: This study aimed to evaluate potential factors that might influence the design of the BLINC device and willingness of participant to undergo the implant placement surgery.
In silico evaluation of corneal patch eluting anti-VEGF agents concept.
Eur J Pharm Biopharm
November 2024
Centre for Advanced Materials and Technologies CEZAMAT, Warsaw University of Technology, Poland. Electronic address:
This study introduces a novel approach utilizing a temporary drug-eluting hydrogel corneal patch to prevent neovascularization, alongside a numerical predictive tool for assessing the release and transport kinetics of bevacizumab (BVZ) after the keratoplasty. A key focus was investigating the impact of tear film clearance on the release kinetics and drug transport from the designed corneal patch. The proposed tear drug clearance model incorporates the physiological mechanism of lacrimal flow (tear turnover), distinguishing itself from previous models.
View Article and Find Full Text PDFThe DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes.
Nat Methods
September 2024
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution fluorescence microscopy technique that achieves single-molecule 'blinking' by transient DNA hybridization. Despite blinking kinetics being largely independent of fluorescent dye choice, the dye employed substantially affects measurement quality. Thus far, there has been no systematic overview of dye performance for DNA-PAINT.
View Article and Find Full Text PDFPhoto-uncaging Triggers on Self-Blinking to Control Single-Molecule Fluorescence Kinetics for Super-resolution Imaging.
ACS Nano
July 2024
State Key Laboratory of Fine Chemicals, Frontiers Science Center for Smart Materials Oriented Chemical Engineering, Dalian University of Technology, Linggong Road 2, Dalian 116024, China.
Super-resolution imaging, especially a single-molecule localization approach, has raised a fluorophore engineering revolution chasing sparse single-molecule dark-bright blinking transforms. Yet, it is a challenge to structurally devise fluorophores manipulating the single-molecule blinking kinetics. In this pursuit, we have developed a triggering strategy by innovatively integrating the photoactivatable nitroso-caging strategy into self-blinking sulfonamide to form a nitroso-caged sulfonamide rhodamine (NOSR).
View Article and Find Full Text PDFMultimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells.
Biomed Opt Express
May 2024
Department of Chemistry, Rice University, 6100 Main St, Houston, TX 77005, USA.
Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!