Photoresponsive Nanocarriers Based on Lithium Niobate Nanoparticles for Harmonic Imaging and On-Demand Release of Anticancer Chemotherapeutics.

Nanoparticle-based drug delivery systems have the potential for increasing the efficiency of chemotherapeutics by enhancing the drug accumulation at specific target sites, thereby reducing adverse side effects and mitigating patient acquired resistance. In particular, photo-responsive nanomaterials have attracted much interest due to their ability to release molecular cargos on demand upon light irradiation. In some settings, they can also provide complementary information by optical imaging on the (sub)cellular scale. We herein present a system based on lithium niobate harmonic nanoparticles (LNO HNPs) for the decoupled multi-harmonic cell imaging and near-infrared light-triggered delivery of an erlotinib derivative (ELA) for the treatment of epidermal growth factor receptor (EGFR)-overexpressing carcinomas. The ELA cargo was covalently conjugated to the surface of silica-coated LNO HNPs through a coumarinyl photo-cleavable linker, achieving a surface loading of the active molecule of 27 nmol/mg NPs. The resulting nanoconjugates (LNO-CM-ELA NPs) were successfully imaged upon pulsed laser excitation at 1250 nm in EGFR-overexpressing human prostate cancer cells DU145 by detecting the second harmonic emission at 625 nm, in the tissue transparency window. Tuning the laser at 790 nm resulted in the uncaging of the ELA cargo as a result of the second harmonic emission of the inorganic HNP core at 395 nm. This protocol induced a significant growth inhibition in DU145 cells, which was only observed upon specific irradiation at 790 nm, highlighting the promising capabilities of LNO-CM-ELA NPs for theranostic applications.

Control of the Two-photon Visual Process in ex vivo Retinas and in Living Mice.

Palcewska et al. first demonstrated near infrared (NIR) visual response in human volunteers upon two-photon absorption (TPA), in a seminal work of 2014, and assessed the process in terms of wavelength- and power-dependence on murine ex-vivo retinas. In the present study, ex-vivo electroretinography (ERG) is further developed to perform a complete characterization of the effect of NIR pulse duration, energy, and focal spot size on the response. The same set of measurements is successively tested on living mice. We discuss how the nonlinear intensity dependence of the photon absorption process is transferred to the amplitude of the visual response acquired by ERG. Finally, we show that the manipulation of the spectral phase of NIR pulses can be translated to predictable change in the two-photon induced response under physiological excitation conditions.

Ultrafast pulse shaping modulates perceived visual brightness in living animals.

Vision is usually assumed to be sensitive to the light intensity and spectrum but not to its spectral phase. However, experiments performed on retinal proteins in solution showed that the first step of vision consists in an ultrafast photoisomerization that can be coherently controlled by shaping the phase of femtosecond laser pulses, especially in the multiphoton interaction regime. The link between these experiments in solution and the biological process allowing vision was not demonstrated. Here, we measure the electric signals fired from the retina of living mice upon femtosecond multipulse and single-pulse light stimulation. Our results show that the electrophysiological signaling is sensitive to the manipulation of the light excitation on a femtosecond time scale. The mechanism relies on multiple interactions with the light pulses close to the conical intersection, like pump-dump (photoisomerization interruption) and pump-repump (reverse isomerization) processes. This interpretation is supported both experimentally and by dynamics simulations.

Circadian hepatocyte clocks keep synchrony in the absence of a master pacemaker in the suprachiasmatic nucleus or other extrahepatic clocks.

It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.

Multiorder Nonlinear Mixing in Metal Oxide Nanoparticles.

Whereas most of the reports on the nonlinear properties of micro- and nanostructures address the generation of distinct signals, such as second or third harmonic, here we demonstrate that the novel generation of dual output lasers recently developed for microscopy can readily increase the accessible parameter space and enable the simultaneous excitation and detection of multiple emission orders such as several harmonics and signals stemming from various sum and difference frequency mixing processes. This rich response, which in our case features 10 distinct emissions and encompasses the whole spectral range from the deep ultraviolet to the short-wave infrared region, is demonstrated using various nonlinear oxide nanomaterials while being characterized and simulated temporally and spectrally. Notably, we show that the response is conserved when the particles are embedded in biological media opening the way to novel biolabeling and phototriggering strategies.

Wavelength-Selective Nonlinear Imaging and Photo-Induced Cell Damage by Dielectric Harmonic Nanoparticles.

We introduce a nonlinear all-optical theranostics protocol based on the excitation wavelength decoupling between imaging and photoinduced damage of human cancer cells labeled by bismuth ferrite (BFO) harmonic nanoparticles (HNPs). To characterize the damage process, we rely on a scheme for in situ temperature monitoring based on upconversion nanoparticles: by spectrally resolving the emission of silica coated NaGdF4:Yb3+/Er3+ nanoparticles in close vicinity of a BFO HNP, we show that the photointeraction upon NIR-I excitation at high irradiance is associated with a temperature increase >100 °C. The observed laser-cell interaction implies a permanent change of the BFO nonlinear optical properties, which can be used as a proxy to read out the outcome of a theranostics procedure combining imaging at 980 nm and selective cell damage at 830 nm. The approach has potential applications to monitor and treat lesions within NIR light penetration depth in tissues.

Author Correction: Preparation from a revisited wet chemical route of phase-pure, monocrystalline and SHG-efficient BiFeO3 nanoparticles for harmonic bio-imaging.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Two-Photon-Triggered Photorelease of Caged Compounds from Multifunctional Harmonic Nanoparticles.

The design of stimuli-responsive nanocarriers has raised much attention to achieve higher local concentration of therapeutics and mitigate the appearance of drug resistance. The combination of imaging properties and controlled photorelease of active molecules within the same nanoconjugate has a great potential for theranostic applications. In this study, a system for NIR light-triggered release of molecular cargos induced by the second harmonic emission from bismuth ferrite harmonic nanoparticles (BFO HNPs) is presented. Silica-coated BFO HNPs were covalently conjugated to a photocaging tether based on coumarin (CM) and l-tryptophan (Trp) as a model molecular cargo. Upon femtosecond pulsed irradiation at 790 nm, Trp was efficiently released from the NP surface in response to the harmonic emission of the nanomaterial at 395 nm. The emitted signal induced the photocleavage of the CM-Trp carbamate linkage resulting in the release of Trp, which was monitored and quantified by ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS). While a small fraction of the uncaging process could be attributed to the nonlinear absorption of CM derivatives, the main trigger responsible for Trp release was established as the second harmonic signal from BFO HNPs. This strategy may provide a new way for the application of functionalized HNPs in dual imaging delivery theranostic protocols.

Bismuth Ferrite Second Harmonic Nanoparticles for Pulmonary Macrophage Tracking.

Recently, second harmonic generation (SHG) nanomaterials have been generated that are efficiently employed in the classical (NIR) and extended (NIR-II) near infrared windows using a multiphoton microscope. The aim was to test bismuth ferrite harmonic nanoparticles (BFO-HNPs) for their ability to monitor pulmonary macrophages in mice. BFO-loaded MH-S macrophages are given intratracheally to healthy mice or BFO-HNPs are intranasally instilled in mice with allergic airway inflammation and lung sections of up to 100 µM are prepared. Using a two-photon-laser scanning microscope, it is shown that bright BFO-HNPs signals are detected from superficially localized cells as well as from deep within the lung tissue. BFO-HNPs are identified with an excellent signal-to-noise ratio and virtually no background signal. The SHG from the nanocrystals can be distinguished from the endogenous collagen-derived SHG around the blood vessels and bronchial structures. BFO-HNPs are primarily taken up by M2 alveolar macrophages in vivo. This SHG imaging approach provides novel information about the interaction of macrophages with cells and the extracellular matrix in lung disease as it is capable of visualizing and tracking NP-loaded cells at high resolution in thick tissues with minimal background fluorescence.

Image Correlation Spectroscopy with Second Harmonic Generating Nanoparticles in Suspension and in Cells.

The absence of photobleaching, blinking, and saturation combined with a high contrast provides unique advantages of higher-harmonic generating nanoparticles over fluorescent probes, allowing for prolonged correlation spectroscopy studies. We apply the coherent intensity fluctuation model to study the mobility of second harmonic generating nanoparticles. A concise protocol is presented for quantifying the diffusion coefficient from a single spectroscopy measurement without the need for separate point-spread-function calibrations. The technique's applicability is illustrated on nominally 56 nm LiNbO3 nanoparticles. We perform label-free raster image correlation spectroscopy imaging in aqueous suspension and spatiotemporal image correlation spectroscopy in A549 human lung carcinoma cells. In good agreement with the expected theoretical result, the measured diffusion coefficient in water at room temperature is (7.5 ± 0.3) µm2/s. The diffusion coefficient in the cells is more than 103 times lower and heterogeneous, with an average of (3.7 ± 1.5) × 10-3 µm2/s.

Preparation from a revisited wet chemical route of phase-pure, monocrystalline and SHG-efficient BiFeO3 nanoparticles for harmonic bio-imaging.

We present two new synthetic routes for bismuth ferrite harmonic nanoparticles (BiFeO3 HNPs). Both phase-pure and mixed phase BiFeO3 materials were produced after improvement of the solvent evaporation and sol-gel combustion routes. Metal nitrates with a series of dicarboxylic acids (tartronic, tartaric and mucic) were used to promote crystallization. We found that the longer the carbon backbone with a hydroxyl group attached to each carbon, the lower the annealing temperature. We also demonstrate that nanocrystals more readily formed at a given temperature by adding glycerol but to the detriment of phase purity, whereas addition of NaCl in excess with mucic acid promotes the formation of phase-pure, monocrystalline nanoparticles. This effect was possibly associated with a better dispersion of the primary amorphous precursors and formation of intermediate complexes. The nanoparticles have been characterized by XRD, TEM, ζ-potential, photon correlation spectroscopy, two-photon microscopy and Hyper-Rayleigh Scattering measurements. The improved crystallization leads to BiFeO3 HNPs without defect-induced luminescence and with a very high averaged second harmonic efficiency (220 pm/V), almost triple the efficiency previously reported. This development of simple, scalable synthesis routes which yield phase-pure and, crucially, monocrystalline BiFeO3 HNPs demonstrates a significant advance in engineering the properties of nanocrystals for bio-imaging and diagnostics applications.

White-Fluorescent Dual-Emission Mechanosensitive Membrane Probes that Function by Bending Rather than Twisting.

Bent N,N'-diphenyl-dihydrodibenzo[a,c]phenazine amphiphiles are introduced as mechanosensitive membrane probes that operate by an unprecedented mechanism, namely, unbending in the excited state as opposed to the previously reported untwisting in the ground and twisting in the excited state. Their dual emission from bent or "closed" and planarized or "open" excited states is shown to discriminate between micelles in water and monomers in solid-ordered (So ), liquid-disordered (Ld ) and bulk membranes. The dual-emission spectra cover enough of the visible range to produce vesicles that emit white light with ratiometrically encoded information. Strategies to improve the bent mechanophores with expanded π systems and auxochromes are reported, and compatibility with imaging of membrane domains in giant unilamellar vesicles by two-photon excitation fluorescence (TPEF) microscopy is demonstrated.

Nonlinear optical susceptibility of two-dimensional WS2 measured by hyper Rayleigh scattering: erratum.

This erratum corrects errors in the expressions for ⟨ßTMD⟩ and fitted form of IHRS and a consequent data point in Fig. 4 of a recent Letter [Opt. Lett.42, 5018 (2017)OPLEDP0146-959210.1364/OL.42.005018]. It also supplies data for the reference compound para-nitroaniline (pNA). The correction to ⟨ßTMD⟩ improves experimental agreement from 46% to within 21% of independent scissors-corrected density functional theory (DFT) calculations. Central findings from the original Letter remain intact.

Bismuth ferrite dielectric nanoparticles excited at telecom wavelengths as multicolor sources by second, third, and fourth harmonic generation.

We demonstrate the simultaneous generation of second, third, and fourth harmonics from a single dielectric bismuth ferrite nanoparticle excited using a telecom fiber laser at 1560 nm. We first characterize the signals associated with different nonlinear orders in terms of spectrum, excitation intensity dependence, and relative signal strengths. Successively, on the basis of the polarization-resolved emission curves of the three harmonics, we discuss the interplay of susceptibility tensor components at different orders and show how polarization can be used as an optical handle to control the relative frequency conversion properties.

Author Correction: Health state dependent multiphoton induced autofluorescence in human 3D in vitro lung cancer model.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Erratum: Publisher's Note: "Implications of short time scale dynamics on long time processes" (Struct. Dyn. 4, 061507 (2017)].

[This corrects the article DOI: 10.1063/1.4996448.].

Nonlinear optical susceptibility of two-dimensional WS2 measured by hyper Rayleigh scattering.

Hyper Rayleigh scattering (HRS) was used to measure the second-order nonlinear susceptibility, χ(2), for liquid exfoliated WS2 monolayers. To the best of our knowledge, it is the first reported application of the HRS technique to assess the bulk-like χ(2) of a two-dimensional (2D) material. The concentration-dependent HRS signal indicated a 4.90±0.30×10-25 esu first hyperpolarizability for 42 nm WS2 monolayers under 1064 nm laser irradiation using para-nitroaniline as an external reference. The corresponding value of χxxx(2) was calculated to be 460±28 pm V-1. This was within 46% of independent density functional theory predictions. Agreement with theory was improved over related microscopy-based approaches. These results support the use of HRS to evaluate 2D materials for nonlinear frequency mixing applications.

Health state dependent multiphoton induced autofluorescence in human 3D in vitro lung cancer model.

Lung diseases pose the highest risk of death and lung cancer is a top killer among cancers with a mortality rate up to 70% within 1 year after diagnosis. Such a fast escalation of this cancer development makes early diagnosis and treatment a highly challenging task, and currently there are no effective tools to diagnose the disease at an early stage. The ability to discriminate between healthy and tumorous tissue has made autofluorescence bronchoscopy a promising tool for detection of lung cancer; however, specificity of this method remains insufficiently low. Here, we perform autofluorescence imaging of human lung cancer invading a human functional airway using an in vitro model of Non Small Cell Lung Cancer which combines a reconstituted human airway epithelium, human lung fibroblasts and lung adenocarcinoma cell lines, OncoCilAir™. By using two-photon laser induced autofluorescence microscopy combined with spectrally resolved imaging, we found that OncoCilAir™ provides tissue's health dependent autofluorescence similar as observed in lung tissue in patients. Moreover, we found spectral and intensity heterogeneity of autofluorescence at the edges of tumors. This metabolic related heterogeneity demonstrates ability of tumor to influence its microenvironment. Together, our result shows that OncoCilAir™ is a promising model for lung cancer research.

Multi-harmonic Imaging in the Second Near-Infrared Window of Nanoparticle-Labeled Stem Cells as a Monitoring Tool in Tissue Depth.

In order to assess the therapeutic potential of cell-based strategies, it is of paramount importance to elaborate and validate tools for monitoring the behavior of injected cells in terms of tissue dissemination and engraftment properties. Here, we apply bismuth ferrite harmonic nanoparticles (BFO HNPs) to in vitro expanded human skeletal muscle-derived stem cells (hMuStem cells), an attractive therapeutic avenue for patients suffering from Duchenne muscular dystrophy (DMD). We demonstrate the possibility of stem cell labeling with HNPs. We also show that the simultaneous acquisition of second- and third-harmonic generation (SHG and THG) from BFO HNPs helps separate their response from tissue background, with a net increase in imaging selectivity, which could be particularly important in pathologic context that is defined by a highly remodelling tissue. We demonstrate the possibility of identifying <100 nm HNPs in depth of muscle tissue at more than 1 mm from the surface, taking full advantage of the extended imaging penetration depth allowed by multiphoton microscopy in the second near-infrared window (NIR-II). Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of hMuStem cells upon exposure to PEG-coated BFO HNPs at different concentrations, revealing their high biocompatibility. Successively, we succeed in detecting individual HNP-labeled hMuStem cells in skeletal muscle tissue after their intramuscular injection.