Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells. Herein, we report the design and development of a small fluorescent molecule, which can be used as an optical probe for G-quadruplexes. We demonstrate that the fluorescence lifetime of this new probe changes considerably upon interaction with different nucleic acid topologies. Specifically, longer fluorescence lifetimes are observed in vitro for G-quadruplexes than for double- and single-stranded nucleic acids. Cellular studies confirm that this molecule is cell permeable, has low cytotoxicity and localizes primarily in the cell nucleus. Furthermore, using fluorescence lifetime imaging microscopy, live-cell imaging suggests that the probe can be used to study the interaction of small molecules with G-quadruplexes in vivo.

Fluorescence Lifetime Imaging (FLIM) is an attractive microscopy method in the life sciences, yielding information on the sample otherwise unavailable through intensity-based techniques. A novel Noise-Corrected Principal Component Analysis (NC-PCA) method for time-domain FLIM data is presented here. The presence and distribution of distinct microenvironments are identified at lower photon counts than previously reported, without requiring prior knowledge of their number or of the dye's decay kinetics. A noise correction based on the Poisson statistics inherent to Time-Correlated Single Photon Counting is incorporated. The approach is validated using simulated data, and further applied to experimental FLIM data of HeLa cells stained with membrane dye di-4-ANEPPDHQ. Two distinct lipid phases were resolved in the cell membranes, and the modification of the order parameters of the plasma membrane during cholesterol depletion was also detected. Noise-corrected Principal Component Analysis of FLIM data resolves distinct microenvironments in cell membranes of live HeLa cells.

In this work, a very simple one-pot synthetic approach was developed to generate aqueous CdTe/CdS/ZnS type-II/type-I red-emitting nanocrystals (NCs). Strain-induced optical properties of CdTe/CdS particles having core (small) /shell (thick) structure with a maximum quantum yield (QY max ) $ 57% were further improved with the overgrowth of a ZnS shell, resulting in a core (small) /shell (thick) /shell (small) structure (QY max $ 64%). The spectral properties were tuned further to the near-infrared region as the ZnS shell grew in thickness. X-ray powder diffraction (XRD) analysis and high-resolution transmission electron microscope (HRTEM) images showed the crystalline structure of NCs proving the epitaxial growth of ZnS without crystalline defects. Under continuous UV-irradiation for 5 h, the NCs did not exhibit any photodegradation but instead displayed a photo-annealing process. These extremely photostable NCs were further characterized in terms of their cytotoxicity and their cell labeling performances. The presence of a ZnS shell was found to reduce the toxicity of the CdTe/CdS NCs. Furthermore, aptamer-conjugated NCs were successfully utilized in targeted cell imaging. Promisingly, the aptamer-NCs bioconjugates were internalized by A549 cells within 2 hours of incubation and retained their fluorescence even after 24 hours of internalization.

Fö rster resonance energy transfer (FRET) is a powerful tool to investigate the interaction between proteins in living cells. Fluorescence proteins, such as the green fluorescent protein (GFP) and its derivatives, are coexpressed in cells linked to proteins of interest. Time-resolved fluorescence anisotropy is a popular tool to study homo-FRET of fluorescent proteins as an indicator of dimerization, in which its signature consists of a very short component at the beginning of the anisotropy decay. In this work, we present an approach to study GFP homo-FRET via a combination of time-resolved fluorescence anisotropy, the stretched exponential decay model, and molecular dynamics simulations. We characterize a new, to our knowledge, FRET standard formed by two enhanced GFPs (eGFPs) and a flexible linker of 15 aminoacids (eGFP15eGFP) with this protocol, which is validated by using an eGFP monomer as a reference. An excellent agreement is found between the FRET efficiency calculated from the fit of the eGFP15eGFP fluorescence anisotropy decays with a stretched exponential decay model (hE exp FRET i ¼ 0.25 5 0.05) and those calculated from the molecular dynamics simulations (hE MD FRET i ¼ 0.18 5 0.14). The relative dipole orientation between the GFPs is best described by the orientation factors hk 2 i ¼ 0.17 5 0.16 and hjk j i ¼ 0.35 5 0.20, contextualized within a static framework in which the linker hinders the free rotation of the fluorophores and excludes certain configurations. The combination of time-and polarization-resolved fluorescence spectroscopy with molecular dynamics simulations is shown to be a powerful tool for the study and interpretation of homo-FRET.

Environmentally-sensitive membrane dyes have been extensively used to study the different liquid phases, (liquid-ordered (Lo) and liquid-disordered (Ld)) of the heterogenous cellular membrane. However, it is not yet well understood how these dyes affect membrane properties upon and post insertion. Using a combination of molecular dynamics (MD) simulations and fluorescence microscopy, we study the effect of PRODAN insertion upon its local environment. We firstly present the results of the MD simulations of PRODAN interacting with lipid bilayers of various compositions, specifically the resultant hydration and lipid order of the system. Experimentally, the lipid order of Lo and Ld vesicles containing various concentrations of PRODAN are inferred from their Generalised Polarisation (GP) values, calculated using their fluorescence spectra. We then apply the methodology to a more complex biological system, the HeLa cell line. For both systems, the presence of PRODAN influences its local environment differently between the Lo and Ld phases. In the simulated systems, the presence of PRODAN lowers the lipid order in the Ld phase and increases the order in the Lo phase, whilst experimental data demonstrates that even a small increase in PRODAN concentration significantly lowers the order of both phases. We suggest this discrepancy may be ascribed to the differing localisations of the dye molecules within the bilayer, and their effect on the hydration of adjacent lipids.

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging ( FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

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The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyancolored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8-(1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMPVenus substrate for thrombin.he most direct and predictable approach to quantify the binding of a target protein is to measure the change in mass or hydrodynamic volume of a fluorescence biosensor following complexation (1). This measurement is most easily achieved by recording the polarized components of fluorescence emission of the probe in the free and bound states, and by computing the fluorescence anisotropy (FA), where:[1]I para and I perp are the fluorescence intensity components parallel and perpendicular to the polarization of the excitation, respectively. The FA value increases in the sensor-target complex owing to the larger hydrodynamic volume and slower rate of tumbling (2). In particular, the Perrin-Weber equation expressed in terms of FA values shows that:where r is the measured FA value and r 0 is the limiting FA value that depends predominantly on the angle between the absorption and emission transition dipole moments, and is in praxis the value of the probe in an extremely viscous solvent (3, 4). τ f is the excitedstate fluorescence lifetime. τ c is the rotational correlation time, the time it takes a sphere to rotate through 1 rad, which is related to the hydrodynamic volume V according to:where η is the solvent viscosity , R is the gas constant, and T is the temperature in Kelvin (3, 4). Eqs. 2 and 3 show that the ideal fluorescent protein probe for FA measurements has a small mass or hydrodynamic volume...