Lifetime Imaging Study Research Articles

Red Circularly Polarized Luminescence from Dimeric H-Aggregates of Acridine Orange by Chiral Induction.

Understanding the mechanism of chirality transfer from a chiral surface to an achiral molecule is essential for designing molecular systems with tunable chiroptical properties. These aspects are explored herein using l- and d-isomers of alkyl valine amphiphiles, which self-assemble in water as nanofibers possessing a negative surface charge. An achiral chromophore, acridine orange, upon electrostatic binding on these surfaces displays mirror-imaged bisignated circular dichroism and red-emitting circularly polarized luminescence signals with a high dissymmetry factor. Experimental and computational investigations establish that the chiroptical properties emerge from surface-bound asymmetric H-type dimers of acridine orange, further supported by fluorescence lifetime imaging studies. Specifically, atomistic molecular dynamics simulations show that the experimentally observed chiral signatures have their origin in van der Waals interactions between acridine orange dimers and the amphiphile head groups as well as in the extent of solvent exposure of the chromophore.

Two-photon excited photoluminescence lifetime imaging studies on individual gelatin-coated gold nanorods

• Intrinsic TPE photoluminescence of different-sized gelatin-coated gold nanorods. • Combined fluorescence spectroscopy and FLIM assays on individual nanorods. • Single gold nanorod TPE photoluminescence overlaps the transverse LSPR band. • Effect of laser excitation power and wavelength on the TPE photoluminescence. The intriguing intrinsic photoluminescence (PL) properties of gold nanorods (AuNRs) have been intensively studied over the last years, not only in the linear but also in the non-linear optics regime, boosting their applicability in various fields of nanobiotechnology. In the present work, we investigate the two-photon excited (TPE) intrinsic PL of different-aspect ratio (AR) synthesized AuNRs, coated with gelatin (g@AuNRs), by performing innovative fluorescence spectroscopy - fluorescence lifetime imaging microscopy (FLIM) combined studies on individual g@AuNRs. In the first step, we employ Finite-difference time-domain (FDTD) simulations to confirm the optical response of AuNRs before and after coating with gelatin. While TPE FLIM assays performed under 800 nm excitation expose some interesting aspect regarding the AR-dependent TPE PL emission generated by individual g@AuNRs, spectroscopic measurements reveal that the TPE PL has a lifetime of around 100 ps and overlaps the corresponding transverse plasmonic band. Finally, we evaluate the effect of laser excitation power and wavelength on the TPE PL, pointing towards the important correlation between these parameters and the TPE PL performance of g@AuNR, as possible contrast agents for label-free TPE fluorescence imaging applications.

Modulation of the Protein-Ligand Interaction in the Presence of Graphene Oxide: a Detailed Spectroscopic Study.

Several applications of graphene oxide (GO) have been established over the years, and it has the potential to be used as a biomedical material. Studying the effect of GO on protein-ligand (small molecules/drugs) complex systems are vital as the mechanisms involved are not well understood. The interaction of GO on the protein-ligand binding is also vital for the preparation of an effective drug carrier in the bloodstream. In this work, we have tried to explore in details the effect of GO on the interaction between a hydrophilic molecule, namely, 7-(N,N'-diethylamino) coumarin-3-carboxylic acid (7-DCA) with human serum albumin (HSA) by employing multispectroscopic, microscopic, calorimetric, and molecular docking studies. We find out that protein-ligand complexes were placed on the GO surface, and GO gives stability to the protein-ligand complex via hydrogen bonding, electrostatic interactions, hydrophobic interactions, and so forth. Due to the presence of a large surface area in GO, it offers a hydrophobic environment, and as a result, the emission maxima of 7-DCA in the ternary complex is more blue-shifted, and the average lifetime becomes higher compared to the binary system. Circular dichroism spectral studies give information about the conformational changes of HSA in the absence and presence of GO when it forms complex with 7-DCA. The fluorescence lifetime imaging study shows the presence of the 7-DCA/HSA complex on the GO sheet. Molecular docking simulation shows that the closest distance between 7-DCA and HSA is 11.9 Å, and the protein interacted with the ligand through hydrogen bonding, hydrophobic interaction, and so forth.

Surface-enhanced fluorescence imaging on linear arrays of plasmonic half-shells.

Here, we perform a Surface-Enhanced Fluorescence (SEF) intensity and lifetime imaging study on linear arrays of silver half-shells (LASHSs), a class of polarization-sensitive hybrid colloidal photonic-plasmonic crystal unexplored previously in SEF. By combining fluorescence lifetime imaging microscopy, scanning confocal fluorescence imaging, Rayleigh scattering imaging, optical microscopy, and finite difference time domain simulations, we identify with high accuracy the spatial locations where SEF effects (intensity increase and lifetime decrease) take place. These locations are the junctions/crevices between adjacent half-shells in the LASHS and locations of high electromagnetic field enhancement and strong emitter-plasmon interactions, as confirmed also by simulated field maps. Such detailed knowledge of the distributed SEF enhancements and lifetime modification distribution, with respect to topography, should prove useful for improved future evaluations of SEF enhancement factors and a more rational design of efficiency-optimized SEF substrates. These linear arrays of metal-coated microspheres expand the family of hybrid colloidal photonic-plasmonic crystals, platforms with potential for applications in optoelectronic devices, fluorescence-based (bio)chemical sensing, or medical assays. In particular, due to the polarized optical response of these LASHSs, specific applications such as hidden tags for anti-counterfeiting or plasmon-enhanced photodetection can be foreseen.

Physical techniques shed light on the differences in sugarcane bagasse structure subjected to steam explosion pretreatments at equivalent combined severity factors

Significant improvements in pretreatments and a deeper understanding of changes in the physical structure and chemical composition of pretreated lignocellulosic materials are required to guarantee the technical and economic sustainability of second-generation ethanol technologies. In present work, several biophysical techniques were applied to characterize sugarcane bagasse samples that have been subjected to autocatalytic, phosphoric acid and sulfuric acid catalyzed steam explosion under equivalent combined severity factors (CSF). Confocal laser scanning microscopy and fluorescence lifetime imaging studies demonstrated unequal changes in lignin distribution in the plant cell wall that could be nicely correlated with substrate susceptibility to enzymatic hydrolysis. Furthermore, solid state nuclear magnetic resonance corroborated these observations by showing the differences in biomass composition primarily associated with hemicellulose removal, whereas X-ray diffraction analysis revealed changes in crystallinity indexes and average crystallite sizes. Thus, the impact of pretreatment temperature on the physical structure and chemical composition of CSF-equivalent steam-exploded sugarcane bagasse was identified as the key factor for developing substrate accessibility. This calls for caution in using combined severity factor as a chief parameter for comparison of different pretreatment conditions.

Polarization-Dependent Photoluminescence of a Highly (100)-Oriented Perovskite Film.

Lead halide perovskite is one of the attractive functional materials owing to its outstanding opto-electronic properties, which have been addressed in numerous studies. This study aims to clarify the link between the growth pattern and the charge carrier related properties for the highly oriented perovskite film along the [100] direction. For this purpose, a CH3 NH3 PbI3 thin film mainly grown along the [100] direction was fabricated and subjected to spectroscopic analysis to understand the basic optoelectronic features of the oriented film. A perovskite film with random growth was also fabricated for comparison. In particular, results from excitation polarization photoluminescence spectroscopy (ExPPS) revealed that the orientation of transition dipole moment, which is relevant to the anisotropic property of the film, is attributed to the growth direction of the perovskite film. This study suggests that the absorption anisotropy can affect the anisotropy in properties of the perovskite device. Furthermore, photodetectors with the perovskite films were fabricated to investigate the effect of growth direction on the photodetector performances, revealing that a photodetector with the oriented perovskite film showed larger photoresponses. In order to provide an explanation for such result, we performed a PL lifetime imaging study of the oriented and randomly grown perovskite films.

Synthesis, Radiolabelling and In Vitro Imaging of Multifunctional Nanoceramics.

Molecular imaging has become a powerful technique in preclinical and clinical research aiming towards the diagnosis of many diseases. In this work, we address the synthetic challenges in achieving lab‐scale, batch‐to‐batch reproducible copper‐64‐ and gallium‐68‐radiolabelled metal nanoparticles (MNPs) for cellular imaging purposes. Composite NPs incorporating magnetic iron oxide cores with luminescent quantum dots were simultaneously encapsulated within a thin silica shell, yielding water‐dispersible, biocompatible and luminescent NPs. Scalable surface modification protocols to attach the radioisotopes 64Cu (t1/2=12.7 h) and 68Ga (t1/2=68 min) in high yields are reported, and are compatible with the time frame of radiolabelling. Confocal and fluorescence lifetime imaging studies confirm the uptake of the encapsulated imaging agents and their cytoplasmic localisation in prostate cancer (PC‐3) cells. Cellular viability assays show that the biocompatibility of the system is improved when the fluorophores are encapsulated within a silica shell. The functional and biocompatible SiO2 matrix represents an ideal platform for the incorporation of 64Cu and 68Ga radioisotopes with high radiolabelling incorporation.

CVD grown 2D MoS2 layers: A photoluminescence and fluorescence lifetime imaging study

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS2. µ‐Raman, µ‐photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS2 films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain‐boundaries. magnified image

Fluorescence lifetime imaging study of a thin protein layer on solid surfaces

Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable medical devices. Fluorescence methods offer the sensitivity required to study the formation of the initial thin protein layers that mediate biocompatibility of materials. Thin protein layers (bovine serum albumin labelled with 1-anilino-8-naphthalenesulfonate, BSA–ANS) deposited on several surfaces (glass, silicon, stainless steel, polystyrene, and silver island film) were studied using confocal frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) and single-point multifrequency lifetime analysis techniques. FLIM provides spatial information about both fluorophores located on the surface and physicochemical parameters of the surface microenvironment. The average fluorescence lifetimes ( τ av) of the adsorbed BSA–ANS generated by the contact between a protein solution and the material surface were measured by the multifrequency modulation and phase shift. Results indicate that τ av values of the albumin complexes on the surfaces (∼ 12 ns) are, in general, shorter than τ av found in the bulk solution (∼ 14 ns). For some surfaces, like polystyrene and silver island film the differences in τ av of the adsorbed BSA–ANS were found to be much greater. The differences in fluorescence lifetimes may indicate structural changes in the BSA protein induced by contact with the surface.

Lipophilic porphyrin microparticles induced by AOT reverse micelles: A fluorescence lifetime imaging study

Fluorescence Lifetime Imaging Microscopy (FLIM) technique was applied to investigate the fluorescence dynamics and structural features of large colloidal aggregates of meso-tetra( N-dodecyl-4-amino sulfonyl-phenyl)porphyrin (PC 12) induced by Sodium 1,4-bis(2-ethyl hexyl)sulfosuccinate (AOT) reverse micelles. The aggregate's particle sizes (down to 1 μm) obtained from the confocal fluorescence images matched with the particle sizes measured in the images obtained from Scanning Electron Microscopy (SEM). The fluorescence decays for those aggregates in the micro spatial domain show triexponential fluorescence lifetimes ( τ 1∼12 ns, τ 2∼3 ns and τ 3∼1 ns) which are independent of the aggregate's size.

AB‐plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies.

NHE1 Regulates the Stratum Corneum Permeability Barrier Homeostasis: MICROENVIRONMENT ACIDIFICATION ASSESSED WITH FLUORESCENCE LIFETIME IMAGING

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.