Single molecule fluorescence burst detection of DNA separated by capillary electrophoresis

1996 ◽  
Author(s):  
Brian B. Haab ◽  
Richard A. Mathies
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Burst Detection ◽  
Single Molecule Fluorescence

Optimization of Single-Molecule Fluorescence Burst Detection of ds-DNA: Application to Capillary Electrophoresis Separations of 100–1000 Basepair Fragments

1997 ◽  
Vol 51 (10) ◽  
pp. 1579-1584 ◽  
Author(s):  
Brian B. Haab ◽  
Richard A. Mathies
Keyword(s):  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Laser Power ◽  
Detection Efficiency ◽  
Fragment Size ◽  
Signal To Noise Ratio ◽  
Burst Size ◽  
Dna Fragments ◽  
Burst Detection ◽  
Single Molecule Fluorescence

Methods for optimizing the dye labeling, laser excitation, and data analysis for single-molecule fluorescence burst detection of ds-DNA have been developed and then validated through capillary electrophoresis (CE) separations of 100–1000 basepair (bp) DNA. Confocal microscopy is used to observe fluorescence bursts from individual DNA fragments labeled with the intercalation dye TO6 as they pass through the ∼ 2-μm-diameter focused laser beam. The dye concentration and laser power were optimized by studying fluorescence burst intensities from pBluescript DNA fragments. The optimal TO6 concentration was ≤100 nM, and the optimal laser power was ≤1 mW. Single-molecule counting was then used to detect CE separations of a 100–1000 bp DNA sizing ladder in 3% linear polyacrylamide. Discrete and baseline-resolved fluorescence bursts were observed in bands as small as 100 bp, and the average burst size within each band increased linearly with fragment size. By counting events using a single optimally chosen discriminator level, we achieve maximum signal-to-noise ratio (S/N) for each fragment size. If the discriminator level is ramped linearly with fragment size to achieve a constant detection efficiency, then the number of events properly reflects the relative fragment concentrations.

scholarly journals Single Molecule Fluorescence Imaging and Blinking

2006 ◽  
Vol 46 (3) ◽  
pp. 164-168
Author(s):  
Hiroaki YOKOTA ◽  
Tetsuichi WAZAWA ◽  
Yoshiharu ISHII
Keyword(s):  
Fluorescence Imaging ◽  
Single Molecule ◽  
Single Molecule Fluorescence

scholarly journals Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

2021 ◽  
Author(s):  
Sujay Ray ◽  
Nibedita Pal ◽  
Nils G Walter
Keyword(s):  
Cluster Analysis ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Holliday Junction ◽  
Energetic Cost ◽  
Genomic Integrity ◽  
Dna Molecules ◽  
Single Molecule Fluorescence ◽  
And Cluster Analysis ◽  
Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

scholarly journals Low-Light Photodetectors for Fluorescence Microscopy

2021 ◽  
Vol 11 (6) ◽  
pp. 2773
Author(s):  
Hiroaki Yokota ◽  
Atsuhito Fukasawa ◽  
Minako Hirano ◽  
Toru Ide
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Single Molecule ◽  
Science Studies ◽  
Single Photon ◽  
Laser Scanning Microscopy ◽  
Oxide Semiconductor ◽  
Wide Field ◽  
Single Molecule Fluorescence ◽  
Low Light

Over the years, fluorescence microscopy has evolved and has become a necessary element of life science studies. Microscopy has elucidated biological processes in live cells and organisms, and also enabled tracking of biomolecules in real time. Development of highly sensitive photodetectors and light sources, in addition to the evolution of various illumination methods and fluorophores, has helped microscopy acquire single-molecule fluorescence sensitivity, enabling single-molecule fluorescence imaging and detection. Low-light photodetectors used in microscopy are classified into two categories: point photodetectors and wide-field photodetectors. Although point photodetectors, notably photomultiplier tubes (PMTs), have been commonly used in laser scanning microscopy (LSM) with a confocal illumination setup, wide-field photodetectors, such as electron-multiplying charge-coupled devices (EMCCDs) and scientific complementary metal-oxide-semiconductor (sCMOS) cameras have been used in fluorescence imaging. This review focuses on the former low-light point photodetectors and presents their fluorescence microscopy applications and recent progress. These photodetectors include conventional PMTs, single photon avalanche diodes (SPADs), hybrid photodetectors (HPDs), in addition to newly emerging photodetectors, such as silicon photomultipliers (SiPMs) (also known as multi-pixel photon counters (MPPCs)) and superconducting nanowire single photon detectors (SSPDs). In particular, this review shows distinctive features of HPD and application of HPD to wide-field single-molecule fluorescence detection.

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