A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.     

Circularly polarized luminescence based on small organic fluorophores

Circularly polarized luminescence (CPL) has attracted attention as a next-generation light signal because of its carrying more information compared with normal and linearly polarized lights as well as its potential wide application in information fields. Recently, much attention has been paid to small organic molecules-based CPL emitters because of easy synthesis, fine structural modification at molecular level, and tunable wide range emission wavelength. This review highlights the development of small organic molecules-based CPL emitters in the past 5 years (2017–2021). The progress suggests that small organic molecules-based CPL emitters provide a simple and efficient way to generate CPL.     

Photocatalytic cleavage of single TiO2/DNA nanoconjugates

TiO(2)/DNA nanoconjugates were successfully fabricated by using the catechol moiety as a binding functional group, which was confirmed by steady-state absorption and fluorescence spectroscopies. Upon UV irradiation, the photocatalytic cleavage of the TiO(2)/DNA nanoconjugates was observed at the single-molecule level by using wide-field fluorescence microscopy. The decrease in the number of conjugates, which was estimated from the luminescent spots due to semiconductor quantum dots modified at the DNA strand, was significantly inhibited by a single A/C mismatch in the DNA sequences. This result strongly suggests that the migration of holes, which are injected from the photoexcited TiO(2) into the DNA, through the DNA bases plays an important role in the photocatalytic cleavage of the conjugates. The influences of the photogenerated reactive oxygen species (ROS) on the cleavage efficiency were also examined. According to the experimental results, it was concluded that oxidation of the catechol moiety and/or the DNA damage are key reactions in this process.     

Ultrafast fluorescence depolarisation in the yellow fluorescent protein due to its dimerisation.

Transient absorption spectroscopy with sub-100 fs time resolution was performed to investigate the oligomerisation behaviour of eYFP in solution. A single time constant tau(AD)=2.2+/-0.15 ps is sufficient to describe the time-resolved anisotropy decay up to at least 200 ps. The close contact of two protein barrels is deduced as the exclusive aggregation state in solution. From the final anisotropy r(infinity)=0.28+/-0.02, the underlying quaternary structure can be traced back to the somewhat distorted structure of the dimers of wt-GFP. The use of autofluorescent proteins as rulers in F?rster resonance energy transfer (FRET) measurements may demand polarisation-sensitive detection of the fluorescence with high time resolution.     

Single carbon nanotube optical spectroscopy.

This Minireview discusses novel insights into the electronic structure of carbon nanotubes obtained using single-molecule fluorescence spectroscopy. Fluorescence spectra from single nanotubes are well described by a single, Lorentzian lineshape. Nanotubes with identical structures fluoresce with different energies due to local electronic perturbations. Carbon nanotube fluorescence unexpectedly does not-show any intensity or spectral fluctuations at 300 K The lack of intensity blinking or bleaching demonstrates that carbon nanotubes have the potential to provide a stable, single-molecule infrared photon source, allowing for the exciting possibility of applications in quantum optics and biophotonics.     

Single-molecule studies of a model fluorenone.

Single-molecule fluorescence measurements of 2,7-bis(3,4,5-trimethoxyphenylethenyl)fluorenone (OFOPV) reveal narrow emission spectra concentrated around 540 nm, with weak emission at longer wavelengths. The wide scattering of emission-maximum wavelengths is attributed to varying molecular environments, with dimers or higher-order aggregates contributing to the low-energy emission. This spectral distribution indicates that emission from monomers of this model fluorenone is mostly green, which is consistent with contaminant emission (g-bands) often observed in fluorene- and polyfluorene-based organic light emitting diode (OLED) devices. A histogram of center wavelengths from 118 single-molecule spectra shows good agreement with the green emission previously observed in thermally stressed 2,7-bis(3,4,5-trimethoxyphenylethenyl)-9,9-diethylfluorene (OFPV). Whereas bulk OFPV exhibits blue fluorescence at about 480 nm, OFOPV bulk thin film measurements reveal red luminescence shifted to 630 nm. This unexpected peak position for bulk OFOPV shifts to higher energies (ca. 540 nm) upon dilution in a solid-state matrix, suggesting that the bulk red emission finds its origins in interactions between fluorenone molecules. Explanations for this red emission include aggregate or excimer formation or intermolecular energy transfer between fluorenone molecules.     

Extraction of kinetic information from single-molecule experiments.

We discuss two possible approaches for extracting kinetic information from single-molecule experiments. The first approach is based on computing correlation functions from measured fluorescence signals, and the second on studying the statistics of on and off times of the same fluorescence signal. We show that in both cases it is possible to extract kinetic information about the nature of intramolecular fluctuations of the single molecule. We show that for single-molecule kinetics the intramolecular fluctuations produce stochastic memory effects which lead to new dynamic features that do not exist in traditional chemical kinetics. In particular, we investigate a new type of chemical oscillations in correlation functions observed experimentally by Edman and Rigler (Proc. Natl. Acad. Sci. USA 2000, 97, 8266).     

Single‐Molecule Electroluminescence of a Phosphorescent Organometallic Complex

Lighting one by one: The electroluminescence (EL) from single molecules of a red phosphorescent iridium complex dispersed in a hole‐transporting polymer matrix is studied. The single‐molecule EL dynamics is determined by local structural inhomogeneities in the matrix polymer (see picture).