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.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

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.

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.

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.

Dynamics of Methylated Cytosine Flipping by UHRF1.

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.

Fluorescence lifetime imaging of membrane lipid order with a ratiometric fluorescent probe.

To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.

Counterion-enhanced cyanine dye loading into lipid nano-droplets for single-particle tracking in zebrafish.

Superior brightness of fluorescent nanoparticles places them far ahead of the classical fluorescent dyes in the field of biological imaging. However, for in vivo applications, inorganic nanoparticles, such as quantum dots, are limited due to the lack of biodegradability. Nano-emulsions encapsulating high concentrations of organic dyes are an attractive alternative, but classical fluorescent dyes are inconvenient due to their poor solubility in the oil and their tendency to form non-fluorescent aggregates. This problem was solved here for a cationic cyanine dye (DiI) by substituting its perchlorate counterion for a bulky and hydrophobic tetraphenylborate. This new dye salt, due to its exceptional oil solubility, could be loaded at 8 wt% concentration into nano-droplets of controlled size in the range 30-90 nm. Our 90 nm droplets, which contained >10,000 cyanine molecules, were >100-fold brighter than quantum dots. This extreme brightness allowed, for the first time, single-particle tracking in the blood flow of live zebrafish embryo, revealing both the slow and fast phases of the cardiac cycle. These nano-droplets showed minimal cytotoxicity in cell culture and in the zebrafish embryo. The concept of counterion-based dye loading provides a new effective route to ultra-bright lipid nanoparticles, which enables tracking single particles in live animals, a new dimension of in vivo imaging.