A practical guide to time-resolved fluorescence microscopy and spectroscopy.

Time-correlated single photon counting (TCSPC) coupled with confocal microscopy is a versatile biophysical tool that enables real-time monitoring of biomolecular dynamics across many timescales. With TCSPC, Fluorescence correlation spectroscopy (FCS) and pulsed interleaved excitation-Förster resonance energy transfer (PIE-FRET) are collected simultaneously on diffusing molecules to extract diffusion characteristics and proximity information. This article is a guide to calibrating FCS and PIE-FRET measurements with several biological samples including liposomes, streptavidin-coated quantum dots, proteins, and nucleic acids for reliable determination of diffusion coefficients and FRET efficiency.

Recurrent mismatch binding by MutS mobile clamps on DNA localizes repair complexes nearby.

DNA mismatch repair (MMR), the guardian of the genome, commences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthesis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL.

Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles.

Coordinated protein and DNA conformational changes govern mismatch repair initiation by MutS.

MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA.

Enhancement of multiphoton emission from single CdSe quantum dots coupled to gold films.

Single molecule time-resolved fluorescence spectroscopy of CdSe/ZnS core-shell quantum dots (QDs) localized near a rough gold thin film demonstrates significant enhancement of multiphoton emission while at the same time showing a decrease in single photon emission. A rigorous analysis of time-resolved photon correlation spectroscopy and fluorescence lifetime data on single quantum dots at room temperature reveals an increase in radiative recombination rate of multiexcitons that is much higher than expected and, perhaps more significantly, is not correlated with concomitant increases in single exciton recombination rates. We believe that these results confirm a stronger coupling of multiexcitons to plasmon modes via a coupling to plasmon multipole modes.