Time-resolved Optical Microscopy Research Articles

The in vitro dynamics of pseudo-vascular network formation

Background/ObjectivesPseudo-vascular network formation in vitro is considered a key characteristic of vasculogenic mimicry. While many cancer cell lines form pseudo-vascular networks, little is known about the spatiotemporal dynamics of these formations.MethodsHere, we present a framework for monitoring and characterising the dynamic formation and dissolution of pseudo-vascular networks in vitro. The framework combines time-resolved optical microscopy with open-source image analysis for network feature extraction and statistical modelling. The framework is demonstrated by comparing diverse cancer cell lines associated with vasculogenic mimicry, then in detecting response to drug compounds proposed to affect formation of vasculogenic mimics. Dynamic datasets collected were analysed morphometrically and a descriptive statistical analysis model was developed in order to measure stability and dissimilarity characteristics of the pseudo-vascular networks formed.ResultsMelanoma cells formed the most stable pseudo-vascular networks and were selected to evaluate the response of their pseudo-vascular networks to treatment with axitinib, brucine and tivantinib. Tivantinib has been found to inhibit the formation of the pseudo-vascular networks more effectively, even in dose an order of magnitude less than the two other agents.ConclusionsOur framework is shown to enable quantitative analysis of both the capacity for network formation, linked vasculogenic mimicry, as well as dynamic responses to treatment.

The supramolecular processing of liposomal doxorubicin hinders its therapeutic efficacy in cells

The successful trajectory of liposome-encapsulated Doxorubicin (e.g. FDA-approved Doxil®) as an anticancer nanodrug in clinical applications is contradicted by in-vitro cell-viability data which highlight its reduced efficacy in promoting cell death compared to non-encapsulated Doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of Doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated Doxorubicin (hereafter ‘L-DOX’) in several in-vitro cellular models. Cell-entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized Doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.

Understanding the Chemical Mechanism behind Photoinduced Enhanced Raman Spectroscopy

Photoinduced enhanced Raman spectroscopy (PIERS) is a new surface enhanced Raman spectroscopy (SERS) modality with a 680% Raman signal enhancement of adsorbed analytes over that of SERS. Despite the explosion in recent demonstrations, the PIERS mechanism remains undetermined. Using X-ray and time-resolved optical spectroscopies, electron microscopy, cyclic voltammetry, and density functional theory simulations, we elucidate the atomic-scale mechanism behind PIERS. Stable PIERS substrates were fabricated using self-organized arrays of TiO2 nanotubes with controlled oxygen vacancy doping and size-controlled silver nanoparticles. The key source of PIERS vs SERS enhancement is an increase in the Raman polarizability of the adsorbed analyte upon photoinduced charge transfer. A balance between improved crystallinity, which enhances charge transfer due to higher electron mobility but decreases light absorption, and increased oxygen vacancy defect concentration, which increases light absorption, is critical. This work enables the rational design of PIERS substrates for sensing.

Impact of Fungal Hyphae on Growth and Dispersal of Obligate Anaerobic Bacteria in Aerated Habitats.

ABSTRACTAnoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes. Here, we analyzed whether and to which degree hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of the obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy, and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches, allowing for spore germination, growth, and fermentative activity of the obligate anaerobe in an otherwise inhabitable environment. Hypoxic liquid films containing 80% ± 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Hyphae hence may serve as good networks for the activity and spatial organization of obligate anaerobic bacteria in oxygenated heterogeneous environments such as soil.

Time-reversal focusing of ultrashort pulses through thin scattering media.

When ultrashort pulses propagate through a disordered medium, scattering occurs and the intensity of the ballistic component decreases drastically. This limits the applicability of time-resolved nonlinear optical spectroscopy and microscopy. The wavefront shaping technique makes it possible to focus light through the scattering medium; however, complete time-reversal of the ultrashort pulses (as short as 10 fs) is still a very challenging problem. This is due to the in-depth characterization and precise control needed for such pulses in the time domain in order to compress down the Fourier-transform limit. In this work, we develop new spatiotemporal wavefront shaping techniques to focus ultrashort pulses at the target position through a thin scattering medium. Compared to other studies, one significant advantage of this method is that most of the characterization of the spectrally-resolved transmission matrix and temporal profile of the ultrashort pulses can be done using single-beam geometry. An interferometer with external reference is necessary to measure the difference of the phase profile between the focused and reference pulses. Furthermore, the number of controllable phase components in the spectral domain is not limited by the spectral correlations of the speckle patterns because we used a pulse shaper in the time domain to optimize the temporal properties of the ultrashort focused pulse. Our new method provides increased flexibility and precise control for manipulating extremely ultrashort pulses through thin scattering media in order to achieve time-reversal focusing at the target position.

Microbubble dynamics and jetting near tissue-phantom biointerfaces.

Precise excitation of cavitation is a promising mechanism for microsurgery procedures and targeted drug delivery enhancement. The underlying phenomenon of interest, jetting behaviour of oscillating cavitation bubbles, occurs due to near-surface interactions between the boundary, liquid, and bubble. Within this study we measured boundary effects on the cavitation bubble dynamics and morphology, with an emphasis on observation and measurement of jetting behaviour near tissue-phantom biointerfaces. An important mechanism of boundary poration has been observed using time-resolved optical microscopy and explained for different tissue-phantom surface densities and Young's modulus. Below a critical distance to the boundary, around γ = 1.0, the resulting jets penetrated the tissue-phantom, resulting in highly localized few micrometer diameter jets.

Investigation of Electronic Quantum Coherence in Semiconductor Materials using Time-resolved Non-Linear Optical Microscopy at Nanoscale Level

Investigation of Electronic Quantum Coherence in Semiconductor Materials using Time-resolved Non-Linear Optical Microscopy at Nanoscale Level

Enhanced Exciton Quantum Coherence in Single CsPbBr3 Perovskite Quantum Dots using Femtosecond Two-Photon Near-Field Scanning Optical Microscopy.

Cesium-halide perovskite quantum dots (QDs) have gained tremendous interest as quantum emitters in quantum information processing applications due to their optical and photophysical properties. However, engineering excitonic states in quantum dots requires a deep knowledge of the coherent dynamics of their excitons at a single-particle level. Here, we use femtosecond time-resolved two-photon near-field scanning optical microscopy (NSOM) to reveal coherences involving a single cesium lead bromide perovskite QD (CsPbBr3) at room temperature. We show that, compared to other nonperovskite nanoparticles, the electronic coherence on a single perovskite QD has a relatively long lifetime of ca. 150 fs, whereas CdSe QDs have exciton coherence times shorter than 75 fs at room temperature. One possible explanation for the longer coherence time observed for the CsPbBr3 perovskite system is related to the exciton fine structure of these perovskite QDs compared to other nanoparticles. These perovskite QDs exhibit interesting optical properties that differ from those of the traditional QDs including bright triplet exciton states. In fact, due to the small amplitude of the energy gap fluctuations of dipole-allowed triplet states in perovskite QDs, the coherent superposition could be preserved for longer times. Furthermore, single-particle excitation approach implemented in this work allows us to remove effects of heterogeneity that are usually present in ensemble averaging experiments at room temperature. The realization of quantum-mechanical phase-coherence of a charge carrier that can operate at room temperature is an issue of great importance for the potential application of coherent electronic phenomena in electronic and optoelectronic devices. These interesting findings provide further evidence of the great potential of these perovskite QDs as candidates for quantum computing and information processing applications.

Discrimination of nano-objects via cluster analysis techniques applied to time-resolved thermo-acoustic microscopy

Time-effective, unsupervised clustering techniques are exploited to discriminate nanometric metal disks patterned on a dielectric substrate. The discrimination relies on cluster analysis applied to time-resolved optical traces obtained from thermo-acoustic microscopy based on asynchronous optical sampling. The analysis aims to recognize similarities among nanopatterned disks and to cluster them accordingly. Each cluster is characterized by a fingerprint time-resolved trace, synthesizing the common features of the thermo-acoustics response of the composing elements. The protocol is robust and widely applicable, not relying on any specific knowledge of the physical mechanisms involved. The present route constitutes an alternative diagnostic tool for on-chip non-destructive testing of individual nano-objects.

Back to the Future: Genetically Encoded Fluorescent Proteins as Inert Tracers of the Intracellular Environment.

Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.

TrLRET microscopy: Ultrasensitive imaging of lanthanide luminophores.

In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.

Suppressing Thermally Induced Fullerene Aggregation in Organic Solar Cells by Employing Plastic Network

Thermally induced fullerene aggregation restricts the long-term stability in organic solar cells. Herein, we demonstrate an effective method of incorporating cross-linkable small-molecule ethoxylated (2) bisphenol-A dimethacrylate (BPA2EODMA) into poly(3-hexylthiophene-2,5-diyl):[6,6]-phenyl C61 butyric acid methyl ester (P3HT:PCBM) blends to form an insoluble framework and inhibit fullerene aggregation. The BPA2EODMA exhibits excellent heat and flexural endurance characteristics, which enhance the thermal and morphological stabilities of the active layer. By systematic research with time-resolved optical spectroscopy and microscopy, the morphological changes and charge-transfer dynamics in P3HT:PCBM blends are explored to unravel the underlying mechanisms for improved photovoltaic efficiency by incorporating the cross-linker. The power conversion efficiency (PCE) increased from 3.6 to 4.2% due to the BPA2EODMA incorporation into the active layer, which is ascribed to the enhancement of exciton dissociati...

Ultrasensitive optical imaging with lanthanide lumiphores.

In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identify three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcome these limitations with a new lanthanide imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.

Time-resolved correlative optical microscopy of charge-carrier transport, recombination, and space-charge fields in CdTe heterostructures

From time- and spatially resolved optical measurements, we show that extended defects can have a large effect on the charge-carrier recombination in II–VI semiconductors. In CdTe double heterostructures grown by molecular beam epitaxy on the InSb (100)-orientation substrates, we characterized the extended defects and found that near stacking faults the space-charge field extends by 2–5 μm. Charge carriers drift (with the space-charge field strength of 730–1,360 V cm−1) and diffuse (with the mobility of 260 ± 30 cm2 V−1 s−1) toward the extended defects, where the minority-carrier lifetime is reduced from 560 ns to 0.25 ns. Therefore, the extended defects are nonradiative recombination sinks that affect areas significantly larger than the typical crystalline grains in II–VI solar cells. From the correlative time-resolved photoluminescence and second-harmonic generation microscopy data, we developed a band-diagram model that can be used to analyze the impact of extended defects on solar cells and other electronic devices.

Thermal melting and ablation dynamics on a femtosecond laser-heated highly-oriented pyrolytic graphite surface

Time-resolved optical reflection microscopy studies demonstrate spatiotemporal dynamics of melting and ablation of graphite surface molten by single IR femtosecond laser pulses, which are revealed by monitoring picosecond oscillations of the probe reflectivity modulated by transient acoustic reverberations in the surface melt. Temporal periods and amplitudes of the reverberations are affected through transient variations of melt thickness and acoustic impedance by melting, thermal expansion, spallation and fragmentation processes, thus enabling quantitative evaluation of their contributions and basic parameters.

Observation of Plasmon Wave Packet Motions via Femtosecond Time-Resolved Near-Field Imaging Techniques.

The generation and dynamics of plasmon wave packets in single gold nanorods were observed at a spatiotemporal scale of 100 nm and 10 fs via time-resolved near-field optical microscopy. Following simultaneous excitation of two plasmon modes of a nanorod with an ultrashort near-field pulse, a decay and revival feature of the time-resolved signal was obtained, which reflected the reciprocating motion of the wave packet. The time-resolved near-field images were also indicative of the wave packet motion. At some period of time after the excitation, the spatial features of the two modes appeared alternately, showing motion of plasmonic wave crests along the rod. The wave packet propagation was clearly demonstrated from this observation with the aid of a simulation model. The present experimental scheme opens the door to coherent control of plasmon-induced optical fields in a nanometer spatial scale and femtosecond temporal scale.

Joule heated metallic microwire devices for sub-microsecond T-jump experiments

We report a study of the transient heating response of submicrometric gold wires on oxidized Si substrates, highlighting their potential as versatile platforms for creating fast temperature variations (T-jumps). To characterize electrically the transient heating response, we developed an original differential resistance setup that excites single wires with current pulses and provides a signal proportional to the ΔT induced. A reproducible sub-microsecond transient heating response (for ΔT≤80°C) was observed for the heaters, regardless the substrate temperature or the presence of a thermal load (polymer film) on the surface. We also developed a simplified mathematical model that reproduced the main experimental observations and provided valuable insights into the dynamics and stability of the heating process. As a proof of concept, our electrical setup was also coupled to a time-resolved optical microscopy setup to detect the transient thermal response of a well-known luminescent thermometer molecule (Rhodamine B).

Plasmon Dephasing in Single Gold Nanorods Observed By Ultrafast Time-Resolved Near-Field Optical Microscopy

We applied time-resolved near-field optical microscopic measurements with ultrashort light pulses of ∼16 fs duration to observe plasmon dephasing processes in single gold nanorods. The correlation widths of the time-resolved signals obtained at each position on the nanorods were broadened compared with the autocorrelation width of the pulse because of the plasmon lifetime. The correlation width maps of the rods showed spatially oscillating patterns that look similar to the plasmon mode structures observed in the static near-field optical images. The spatial variation of the correlation widths was explained as arising from the position-dependent contribution of the resonant plasmon excitation in the time-resolved signals relative to that of the nonresonant excitation. This finding indicates that the dephasing times of the resonant plasmon modes were constant regardless of the excitation position. This result is understood to be a consequence of the spatial coherence of the plasmon mode that causes the loca...

Silicon as a virtual plasmonic material: Acquisition of its transient optical constants and the ultrafast surface plasmon-polariton excitation

Ultrafast intense photoexcitation of a silicon surface is complementarily studied experimentally and theoretically, with its prompt optical dielectric function obtained by means of time-resolved optical reflection microscopy and the underlying electron-hole plasma dynamics modeled numerically, using a quantum kinetic approach. The corresponding transient surface plasmon-polariton (SPP) dispersion curves of the photo-excited material were simulated as a function of the electron-hole plasma density, using the derived optical dielectric function model, and directly mapped at several laser photon energies, measuring spatial periods of the corresponding SPP-mediated surface relief nanogratings. The unusual spectral dynamics of the surface plasmon resonance, initially increasing with the increase in the electron-hole plasma density but damped at high interband absorption losses induced by the high-density electron-hole plasma through instantaneous bandgap renormalization, was envisioned through the multi-color mapping.

Directional wetting in anisotropic inverse opals.

Porous materials display interesting transport phenomena due to restricted motion of fluids within the nano- to microscale voids. Here, we investigate how liquid wetting in highly ordered inverse opals is affected by anisotropy in pore geometry. We compare samples with different degrees of pore asphericity and find different wetting patterns depending on the pore shape. Highly anisotropic structures are infiltrated more easily than their isotropic counterparts. Further, the wetting of anisotropic inverse opals is directional, with liquids filling from the side more easily. This effect is supported by percolation simulations as well as direct observations of wetting using time-resolved optical microscopy.