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.