Is Single-Molecule Fluorescence Spectroscopy Ready To Join the Organic Chemistry Toolkit? A Test Case Involving Click Chemistry

Single molecule spectroscopy (SMS) has matured to a point where it can be used as a convenient tool to guide organic synthesis and drug discovery, particularly applicable to catalytic systems where questions related to homogeneous vs heterogeneous pathways are important. SMS can look at intimate mechanistic details that can inspire major improvements of the catalyst performance, its recovery, and reuse. Here, we use the click reaction between alkynes and azides as an example where improvements at the bench have been inspired and validated using single-molecule fluorescence spectroscopy

Detection of Single Ion Spectra by Coulomb Crystal Heating

The coupled motion of ions in a radiofrequency trap has been used to connect the frequency- dependent laser-induced heating of a sympathetically cooled spectroscopy ion with changes in the fluorescence of a laser-cooled control ion. This technique, sympathetic heating spectroscopy, is demonstrated using two isotopes of calcium. In the experiment, a few scattered photons from the spectroscopy ion are transformed into a large deviation from the steady-state fluorescence of the control ion. This allows us to detect an optical transition where the number of scattered photons is below our fluorescence detection limit. Possible applications of the technique to molecular ion spectroscopy are briefly discussed.Comment: 7 Pages,10 Figure

Polymer dynamics, fluorescence correlation spectroscopy, and the limits of optical resolution

In recent years, fluorescence correlation spectroscopy has been increasingly applied for the study of polymer dynamics on the nanometer scale. The core idea is to extract, from a measured autocorrelation curve, an effective mean-square displacement function that contains information about the underlying conformational dynamics. The paper presents a fundamental study of the applicability of fluorescence correlation spectroscopy for the investigation of nanoscale conformational and diffusional dynamics. We find that fluorescence correlation spectroscopy cannot reliably elucidate processes on length scales much smaller than the resolution limit of the optics used and that its improper use can yield spurious results for the observed dynamics.Comment: 4 pages, 4 figures, accepted by Physical Review Letter

Plasmonic antennas and zero mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy towards physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as F\"orster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero mode waveguides and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometre scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET and FCS. Single molecule spectroscopy techniques greatly benefit from zero mode waveguides and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.Comment: WIREs Nanomed Nanobiotechnol 201

Exploratory analysis of excitation-emission matrix fluorescence spectra with self-organizing maps as a basis for determination of organic matter removal efficiency at water treatment works

In the paper, the self-organizing map (SOM) was employed for the exploratory analysis of fluorescence excitation-emission data characterizing organic matter removal efficiency at 16 water treatment works in the UK. Fluorescence spectroscopy was used to assess organic matter removal efficiency between raw and partially treated (clarified) water to provide an indication of the potential for disinfection by-products formation. Fluorescence spectroscopy was utilized to evaluate quantitative and qualitative properties of organic matter removal. However, the substantial amount of fluorescence data generated impeded the interpretation process. Therefore a robust SOM technique was used to examine the fluorescence data and to reveal patterns in data distribution and correlations between organic matter properties and fluorescence variables. It was found that the SOM provided a good discrimination between water treatment sites on the base of spectral properties of organic matter. The distances between the units of the SOM map were indicative of the similarity of the fluorescence samples and thus demonstrated the relative changes in organic matter content between raw and clarified water. The higher efficiency of organic matter removal was demonstrated for the larger distances between raw and clarified samples on the map. It was also shown that organic matter removal was highly dependent on the raw water fluorescence properties, with higher efficiencies for higher emission wavelengths in visible and UV humic-like fluorescence centers

Examination of 4’,6-diamidino-2-phenylindole (DAPI) in Silica Gels through Fluorometry

Silica sol-gels synthesized through hydrolysis and condensation reactions via acid- and base-catalyzed procedures containing 4’,6-diamidino-2-phenylindole (DAPI) have been examined using fluorescence spectroscopy. DAPI is a fluorescent molecule that has traditionally been used in biosensors as a target molecule and a fluorescent stain known to bind strongly to the A-T rich regions of DNA. Sol-gels containing various concentrations of DAPI were dried conventionally to form xerogels or supercritically to form aerogels and then analyzed using fluorescence spectroscopy to determine the most optimal concentration of DAPI

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactiv