Alix Le Marois

The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy

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.

In vivo biodistribution studies and ex vivo lymph node imaging using heavy metal-free quantum dots

Quantum dots (QDs) are attractive photoluminescence probes for biomedical imaging due to their unique photophysical properties. However, the potential toxicity of QDs has remained a major obstacle to their clinical use because they commonly incorporate the toxic heavy metal cadmium within the core of the QDs. In this work, we have evaluated a novel type of heavy metal-free/cadmium-free and biocompatible QD nanoparticles (bio CFQD® nanoparticles) with a good photoluminescence quantum yield. Sentinel lymph node mapping is an increasingly important treatment option in the management of breast cancer. We have demonstrated their potential for lymph node mapping by ex vivo imaging of regional lymph nodes after subcutaneous injection in the paw of rats. Using photoluminescence imaging and chemical extraction measurements based on elemental analysis by inductively coupled plasma mass spectroscopy, the quantum dots are shown to accumulate quickly and selectively in the axillary and thoracic regional lymph nodes. In addition, lifetime imaging microscopy of the QD photoluminescence indicates minimal perturbation to their photoluminescence properties in biological systems.

One-pot aqueous synthesis of highly strained CdTe/CdS/ZnS nanocrystals and their interactions with 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 (QYmax) ∼ 57% were further improved with the overgrowth of a ZnS shell, resulting in a core(small)/shell(thick)/shell(small) structure (QYmax ∼ 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 photo-degradation 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.

Genetically encoded sensors of protein hydrodynamics and molecular proximity.

Significance The lumazine binding protein (LUMP) emits a cyan-colored fluorescence and has the longest average fluorescence lifetime of any genetically encoded fluorescent protein complex. Coupled with a small mass of 20 kDa, LUMP and its fusion with capture sequences are exploited as unique sensors of protein hydrodynamics and are shown to enable quantitative fluorescence anisotropy imaging of specific target proteins in vitro and in vivo. Moreover, the surface location of the lumazine probe is shown to improve the efficiency of Förster resonance energy transfer (FRET) with the Venus acceptor protein compared with CFP, which is used in the development of a new class of FRET-based sensor. 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 cyan-colored 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 LUMP-Venus substrate for thrombin.

Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reduced lifetime near the coverslip in TIR compared to epifluorescence FLIM.

Noise-Corrected Principal Component Analysis of fluorescence lifetime imaging data.

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 dyes 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.

Wide-field TCSPC-based fluorescence lifetime imaging (FLIM) microscopy

Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.

Quantitative Live Cell FLIM Imaging in Three Dimensions

In this chapter, the concept of fluorescence lifetime and its utility in quantitative live cell imaging will be introduced, along with methods to record and analyze FLIM data. Relevant applications in 3D tissue and live cell imaging, including multiplexed FLIM detection, will also be detailed.

Fluorescence Lifetime Imaging (FLIM): Basic Concepts and Recent Applications

Fluorescence lifetime imaging (FLIM) is a key fluorescence microscopy technique to map the environment and interaction of fluorescent probes. It can report on photo physical events that are difficult or impossible to observe by fluorescence intensity imaging, because FLIM is independent of the local fluorophore concentration and excitation intensity. A FLIM application relevant for biology concerns the identification of FRET to study protein interactions and conformational changes, and FLIM can also be used to image viscosity, temperature, pH, refractive index and ion and oxygen concentrations, all at the cellular or sub-cellular level, as well as autofluorescence. The basic principles and some recent advances in the application of FLIM, FLIM instrumentation and molecular probe development will be discussed.

PRODAN differentially influences its local environment

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.