Thousand-fold Enhancement of Single-Molecule Fluorescence Near a Single Gold Nanorod

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.

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Silicon Microchannel-Driven Raman Scattering Enhancement to Improve Gold Nanorod Functions as a SERS Substrate toward Single-Molecule Detection

ACS Applied Materials & Interfaces ◽

10.1021/acsami.1c08480 ◽

2021 ◽

Author(s):

Jaciara Bär ◽

Anerise de Barros ◽

Davi H. S. de Camargo ◽

Mariane P. Pereira ◽

Leandro Merces ◽

...

Keyword(s):

Raman Scattering ◽

Single Molecule ◽

Gold Nanorod ◽

Single Molecule Detection ◽

Sers Substrate

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Low-Light Photodetectors for Fluorescence Microscopy

Applied Sciences ◽

10.3390/app11062773 ◽

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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