Time-resolved Fluorescence Anisotropy Imaging Research Articles

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy.

Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, imaging is still rarely investigated in general and in carbon dots (CDs) in particular. To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and , together with the steady-state images of fluorescence intensity (FI) and FA (). The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. The FLT of fluorescein samples was found to decrease from to , whereas both and were significantly increased from to and to , respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of from to and from to for the first CDs and from to and to for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, , and ). Nevertheless, was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano-environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano-environments in complex, biological systems.

Photophysical and Fluorescence Anisotropic Behavior of Polyfluorene β-Conformation Films.

We demonstrate a systematic visualization of the unique photophysical and fluorescence anisotropic properties of polyfluorene coplanar conformation (β-conformation) using time-resolved scanning confocal fluorescence imaging (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) measurements. We observe inhomogeneous morphologies and fluorescence decay profiles at various micrometer-sized regions within all types of polyfluorene β-conformational spin-coated films. Poly(9,9-dioctylfluorene-2,7-diyl) (PFO) and poly[4-(octyloxy)-9,9-diphenylfluoren-2,7-diyl]-co-[5-(octyloxy)-9,9-diphenylfluoren-2,7-diyl] (PODPF) β-domains both have shorter lifetime than those of the glassy conformation for the longer effective conjugated length and rigid chain structures. Besides, β-conformational regions have larger fluorescence anisotropy for the low molecular rotational motion and high chain orientation, while the low anisotropy in glassy conformational regions shows more rotational freedom of the chain and efficient energy migration from amorphous regions to β-conformation as a whole. Finally, ultrastable ASE threshold in the PODPF β-conformational films also confirms its potential application in organic lasers. In this regard, FLIM and FAIM measurements provide an effective platform to explore the fundamental photophysical process of conformational transitions in conjugated polymer.

Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging

Neural activity relies on molecular diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is critical for understanding the dynamics of molecular events and electric currents that shape physiological signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small molecules move on average ~30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~70%. In the synaptic cleft free nanodiffusion is decelerated by ~46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the electrical conductance of the brain interstitial space and the limiting rate of molecular interactions or conformational changes in the synaptic microenvironment.

Imaging Supported Interfacial Layers – Factors that Influence Interface Organization and Dynamics

Supported interfacial structures are centrally important to a variety of electrochemical and chemical sensing applications. Lipid bilayer structures have received widespread attention not only because of their potential utility in creating biomimetic devices but also because of their inherent complexity and sensitivity to their environment. Understanding the factors that influence organization and dynamics in such systems could be of immediate relevance to the chemical sensing and biological communities. Our specific interest lies in the formation of interfaces that could be used to host transmembrane proteins for sensing applications. Knowledge of the relationships between bilayer composition and morphology, as well as the influence of overlayer constituents, is a necessary first step in the creation of useful supported bilayer interfaces. We have constructed model bilayer structures containing phosphocholine, sphingomyelin and cholesterol supported on mica, and have examined the organization of and dynamics within these phase-segregated structures using time-resolved fluorescence lifetime and anisotropy imaging. Our results show that these supported bilayer structures cannot be explained simply in the context of phase segregation of cholesterol and phosphocholine domains, and that the organization of these bilayers depends sensitively on the constituents present in liquid overlayers. Our image and dynamic data provide a useful starting point for the construction of biomimetic bilayer structures capable of housing biomolecules for sensing applications.

Time-resolved fluorescence anisotropy imaging.

Fluorescence can be characterized by its intensity, position, wavelength, lifetime, and polarization. The more of these features are acquired in a single measurement, the more can be learned about the sample, i.e., the microenvironment of the fluorescence probe. Polarization-resolved fluorescence lifetime imaging-time-resolved fluorescence anisotropy imaging, TR-FAIM-allows mapping of viscosity or binding or of homo-FRET which can indicate dimerization or generally oligomerization.

Homodimerization of Amyloid Precursor Protein at the Plasma Membrane: A homoFRET Study by Time-Resolved Fluorescence Anisotropy Imaging

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented. The correcting factor accounting for the depolarization due to the high numerical aperture (NA) objective, mandatory for TIRF microscopy, was quantified on fluorescein solutions and on HEK293 cells expressing enhanced Green Fluorescence Protein (eGFP). Homodimerization of Amyloid Precursor Protein (APP), a key mechanism in the etiology of Alzheimer’s disease, was measured on this original set-up. We showed, both in epifluorescence and under TIRF excitation, different energy transfer rates associated with the homodimerization of wild type APP-eGFP or of a mutated APP-eGFP, which forms constitutive dimers. This original set-up thus offers promising prospects for future studies of protein homodimerization in living cells in control and pathological conditions.

Mapping viscosity in living cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes, and viscosity is one of the key parameters affecting transport of molecules and proteins. In biological specimen, changes in viscosity have been linked to disease and malfunction at the cellular level. Viscosity also plays a role in drug delivery and cancer therapy. While methods to measure the bulk viscosity are well developed, they require macroscopic sample quantities and use mechanical or fluid-dynamics approaches.1 However, microscale measurements remain a challenge. Until recently, viscosity maps of single cells have been hard to obtain. Optical techniques are powerful tools for studying biological samples: since they are nondestructive and minimally invasive, they can be used with living cells and tissues.2, 3 Measurements can be made in situ, thus allowing access to biological function within a true physiological context. Fluorescence-imaging techniques, in particular, are widely used because of their high contrast and easy visualization of proteins and their cellular environment. The relevant detection sensitivity extends down to the single-molecule level. Moreover, fluorescence can be characterized by multiple parameters, including intensity, wavelength, lifetime, and polarization. In particular, polarization-resolved lifetime measurements allow scientists to determine the rotational mobility of fluorophores.4 This approach depends on the viscosity of their surroundings. Thus, polarization-resolved fluorescence lifetime imaging (FLIM) or time-resolved fluorescence anisotropy imaging (TR-FAIM) can be used to map viscosity.5, 6 We recently demonstrated the feasibility of using nanosecond FLIM of molecular rotors to image microviscosity in living cells.7–9 Their structures are shown in Figure 1. Molecular rotors have a fluorescence lifetime that varies with the viscosity of their micro-environment.10 In contrast to TR-FAIM, FLIM requires no polarization-resolved detection of the fluorescence emission (or polarized excitation). The fluorescence lifetime can directly be converted into a viscosity with a calibration based on the Forster Hoffmann model.9 Figure 1. (a) and (b) Boron dipyrromethene (bodipy)9 and (c) porphyrin-based8 molecular rotors.

Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy

A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy r(inf) was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.

Time-resolved fluorescence microscopy

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.

Time-resolved fluorescence anisotropy imaging applied to live cells.

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

We report a picosecond time-gated fluorescence lifetime imaging (FLIM) system extended to perform time-resolved fluorescence anisotropy imaging (TR-FAIM). Upon excitation with linearly polarized laser pulses, the parallel and perpendicular components of the fluorescence emission from a sample are imaged simultaneously using a polarization-resolved imager. The imaging technique presented here quantitatively reports the rotational mobility of a fluorophore as it varies according to the local environment. In a single acquisition run it yields maps of both rotational correlation time and fluorescence lifetime as they vary across a sample. TR-FAIM has been applied to imaging standard multiwell plate samples of rhodamine 6G dissolved in methanol, ethylene glycol, trimethylene glycol, and glycerol. The observed rotational correlation times and fluorescence lifetimes, which report the local viscosity and refractive index of the local rhodamine 6G environment, respectively, are in good agreement with previously published single point measurements. By considering the linear dependence of the rotational correlation time on viscosity up to 20 cP, we are able to obtain a two-dimensional viscosity map. Wide-field maps of rotational correlation time, and therefore viscosity, have been obtained. This illustrates the potential to image the local viscosity and fluorescence lifetime distributions of fluorophore tagged proteins in cells.