Luminescent Iridium-Peptide Nucleic Acid Bioconjugate as Photosensitizer for Singlet Oxygen Production toward a Potential Dual Therapeutic Agent.

A novel bioorganometallic PNA conjugate () was synthesized by covalently bonding a model PNA tetramer to a luminescent bis-cyclometalated Ir(III) complex that acted as a photosensitizer under light irradiation to generate singlet oxygen (O). The conjugate was prepared using an Ir complex bearing the 1,10-phenanthroline ligand functionalized with either a free primary amine () or a carboxyl group () for the conjugation to PNA. The photophysical studies on the and the demonstrated that the luminescent properties were maintained after the conjugation of the Ir fragment to PNA.

How Tumors Affect Hemodynamics: A Diffusion Study on the Zebrafish Transplantable Model of Medullary Thyroid Carcinoma by Selective Plane Illumination Microscopy.

Medullary thyroid carcinoma (MTC), a rare neuroendocrine tumor comprising 3-5% of thyroid cancers, arises from calcitonin-producing parafollicular C cells. Despite aggressive behavior, surgery remains the primary curative treatment, with limited efficacy reported for radiotherapy and chemotherapy. Recent efforts have explored the pathogenetic mechanisms of MTC, identifying it as a highly vascularized neoplasm overexpressing pro-angiogenic factors.

Thiolated Cyanines Appended to Partially Pegylated Gold Nanoparticles for Fluorescence Quenching of Two-Channel Photothermal Inks.

Following a new approach, we prepared a nanoink with two separate photothermally responsive absorption bands. One is the localized surface plasmon resonance (LSPR) absorption of gold nanoparticles (AuNP, d=17 nm), the second is the absorption band of two cyanine (Cy) dyes, Cy7-C6 or Cy7-C11, grafted to the AuNP surface through thiolated bridges of different lengths: the close proximity to the Au surface induces full quenching of the Cy fluorescence, resulting in thermal relaxation on irradiation. Attempts to full coat AuNP with the lipophilic Cy7-C6 and Cy7-C11 lead to precipitation from aqueous solutions.

Proteinaceous microstructure in a capillary: a study of non-linear bending dynamics.

The flap of bendable structures under continuous flow impacts a variety of fields, ranging from energy harvesting to active mixing in microfluidic devices. Similar physical principles determine the flapping dynamics in a variety of systems with different sizes, but a thorough investigation of the bending dynamics at the microscale is still lacking. We employ here two-photon laser polymerization to fabricate elongated proteinaceous flexible microstructures directly within a micro-capillary and we characterize their bending dynamics.

Quantitative active super-resolution thermal imaging: The melanoma case study.

Super-resolution image acquisition has turned photo-activated far-infrared thermal imaging into a promising tool for the characterization of biological tissues. By the sub-diffraction localization of sparse temperature increments primed by the sample absorption of modulated focused laser light, the distribution of (endogenous or exogenous) photo-thermal biomarkers can be reconstructed at tunable ∼10-50 μm resolution. We focus here on the theoretical modeling of laser-primed temperature variations and provide the guidelines to convert super-resolved temperature-based images into quantitative maps of the absolute molar concentration of photo-thermal probes.

Melanin concentration maps by label-free super-resolution photo-thermal imaging on melanoma biopsies.

Surgical excision followed by histopathological examination is the gold standard for melanoma screening. However, the color-based inspection of hematoxylin-and-eosin-stained biopsies does not provide a space-resolved quantification of the melanin content in melanocytic lesions. We propose a non-destructive photo-thermal imaging method capable of characterizing the microscopic distribution and absolute concentration of melanin pigments in excised melanoma biopsies.

Multiphoton Laser Fabrication of Hybrid Photo-Activable Biomaterials.

The possibility to shape stimulus-responsive optical polymers, especially hydrogels, by means of laser 3D printing and ablation is fostering a new concept of "smart" micro-devices that can be used for imaging, thermal stimulation, energy transducing and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. In light of the increasing demand for micro-devices for nanomedicine and personalized medicine, interest is growing in the combination of composite and hybrid photo-responsive materials and digital micro-/nano-manufacturing.

PVA Films with Mixed Silver Nanoparticles and Gold Nanostars for Intrinsic and Photothermal Antibacterial Action.

PVA films with embedded either silver nanoparticles (AgNP), NIR-absorbing photothermal gold nanostars (GNS), or mixed AgNP+GNS were prepared in this research. The optimal conditions to obtain stable AgNP+GNS films with intact, long lasting photothermal GNS were obtained. These require coating of GNS with a thiolated polyethylene glycol (PEG) terminated with a carboxylic acid function, acting as reticulant in the film formation.

Photothermally active nanoparticles as a promising tool for eliminating bacteria and biofilms.

Bacterial contamination is a severe issue that affects medical devices, hospital tools and surfaces. When microorganisms adhere to a surface (e.g.

Nanocomposite Sprayed Films with Photo-Thermal Properties for Remote Bacteria Eradication.

Currently there is a strong demand for novel protective materials with efficient antibacterial properties. Nanocomposite materials loaded with photo-thermally active nanoparticles can offer promising opportunities due to the local increase of temperature upon near-infrared (NIR) light exposure capable of eradicating bacteria. In this work, we fabricated antibacterial films obtained by spraying on glass slides aqueous solutions of polymers, containing highly photo-thermally active gold nanostars (GNS) or Prussian Blue (PB) nanoparticles.

Self-Assembled Monolayers of Copper Sulfide Nanoparticles on Glass as Antibacterial Coatings.

We developed an easy and reproducible synthetic method to graft a monolayer of copper sulfide nanoparticles (CuS NP) on glass and exploited their particular antibacterial features. Samples were fully characterized showing a good stability, a neat photo-thermal effect when irradiated in the Near InfraRed (NIR) region (in the so called "biological window"), and the ability to release controlled quantities of copper in water. The desired antibacterial activity is thus based on two different mechanisms: (i) slow and sustained copper release from CuS NP-glass samples, (ii) local temperature increase caused by a photo-thermal effect under NIR laser irradiation of CuS NP-glass samples.

Whole-Section Tumor Micro-Architecture Analysis by a Two-Dimensional Phasor-Based Approach Applied to Polarization-Dependent Second Harmonic Imaging.

Second Harmonic Generation (SHG) microscopy has gained much interest in the histopathology field since it allows label-free imaging of tissues simultaneously providing information on their morphology and on the collagen microarchitecture, thereby highlighting the onset of pathologies and diseases. A wide request of image analysis tools is growing, with the aim to increase the reliability of the analysis of the huge amount of acquired data and to assist pathologists in a user-independent way during their diagnosis. In this light, we exploit here a set of phasor-parameters that, coupled to a 2-dimensional phasor-based approach (μMAPPS, Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG signal) and a clustering algorithm, allow to automatically recover different collagen microarchitectures in the tissues extracellular matrix.

Novel photo-thermally active polyvinyl alcohol-Prussian blue nanoparticles hydrogel films capable of eradicating bacteria and mitigating biofilms.

Antibacterial treatment is an essential issue in many diverse fields, from medical device treatments (for example prostheses coating) to food preservation. However, there is a need of novel and light-weight materials with high antibacterial efficiency (preferably due to the physical activation). Utilization of photo-thermally active nanoparticles can lead to novel and re-usable materials that can be remotely activated on-demand to thermally eradicate bacteria and mitigate biofilm formation, therefore meeting the above challenge.

Adaptive optics microspectrometer for cross-correlation measurement of microfluidic flows.

Mapping flows in vivo is essential for the investigation of cardiovascular pathologies in animal models. The limitation of optical-based methods, such as space-time cross correlation, is the scattering of light by the connective and fat components and the direct wave front distortion by large inhomogeneities in the tissue. Nonlinear excitation of the sample fluorescence helps us by reducing light scattering in excitation.

Spatiotemporal Image Correlation Analysis for 3D Flow Field Mapping in Microfluidic Devices.

Microfluidic devices reproducing 3D networks are particularly valuable for nanomedicine applications such as tissue engineering and active cell sorting. There is however a gap in the possibility to measure how the flow evolves in such 3D structures. We show here that it is possible to map 3D flows in complex microchannel networks by combining wide field illumination to image correlation approaches.

Spatiotemporal image correlation analysis of blood flow in branched vessel networks of zebrafish embryos.

Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images.

Synthesis of reduced-size gold nanostars and internalization in SH-SY5Y cells.

The synthesis of large pentatwinned five-branched gold nanostars (GNS) has been modified so to obtain overall dimensions shrunk to 60% and a lower branches aspect ratio, leading to a dramatic blue shift of their two near-infrared (NIR) localized surface plasmon resonances (LSPR) absorptions but still maintaining one LSPR in the biotransparent NIR range. The interactions of polyethylene glycol (PEG) coated large and shrunk GNS with SH-SY5Y cells revealed that the large ones (DCI - diameter of the circumference in which GNS can be inscribed=76nm) are internalized more efficiently than the shrunk ones (DCI=46nm), correlating with a decreased cells surviving fraction.

Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes.

In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells.

An Intermittent Model for Intracellular Motions of Gold Nanostars by k-Space Scattering Image Correlation.

Anisotropic metallic nanoparticles have been devised as powerful potential tools for in vivo imaging, photothermal therapy, and drug delivery thanks to plasmon-enhanced absorption and scattering cross sections, ease in synthesis and functionalization, and controlled cytotoxicity. The rational design of all these applications requires the characterization of the nanoparticles intracellular trafficking pathways. In this work, we exploit live-cell time-lapse confocal reflectance microscopy and image correlation in both direct and reciprocal space to investigate the intracellular transport of branched gold nanostars (GNSs).

Monolayers of gold nanostars with two near-IR LSPRs capable of additive photothermal response.

Monolayers of photothermally responsive gold nanostars on PEI-coated surfaces display two localized surface plasmon resonances (LSPRs) in the near-IR region that can be laser-irradiated either separately, obtaining two different T jumps, or simultaneously, obtaining a T jump equal to the sum of those obtained with separate irradiations.

Thermal and Chemical Stability of Thiol Bonding on Gold Nanostars.

The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose.

Gold nanostars coated with neutral and charged polyethylene glycols: A comparative study of in-vitro biocompatibility and of their interaction with SH-SY5Y neuroblastoma cells.

Gold nanostars (GNS) have been coated with four different polyethylene glycols (PEGs) equipped with a -SH function for grafting on the gold surface. These PEGs have different chain lengths with average MW=2000, 3000, 5000 and average number of -O-CH2-CH2 - units 44, 66, and 111, respectively. Two are neutral and two are terminated with -COOH and -NH2 functions, thus bearing negative and positive charges at physiological pH, thanks to the formation of carboxylate and ammonium groups.

A luminescent poly(amidoamine)-iridium complex as a new singlet-oxygen sensitizer for photodynamic therapy.

A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water.

In vivo flow mapping in complex vessel networks by single image correlation.

We describe a novel method (FLICS, FLow Image Correlation Spectroscopy) to extract flow speeds in complex vessel networks from a single raster-scanned optical xy-image, acquired in vivo by confocal or two-photon excitation microscopy. Fluorescent flowing objects produce diagonal lines in the raster-scanned image superimposed to static morphological details. The flow velocity is obtained by computing the Cross Correlation Function (CCF) of the intensity fluctuations detected in pairs of columns of the image.

Electron multiplying charge-coupled device-based fluorescence cross-correlation spectroscopy for blood velocimetry on zebrafish embryos.

Biomedical issues in vasculogenesis and cardiogenesis require methods to follow hemodynamics with high spatial (micrometers) and time (milliseconds) resolution. At the same time, we need to follow relevant morphogenetic processes on large fields of view. Fluorescence cross-correlation spectroscopy coupled to scanning or wide-field microscopy meets these needs but has limited flexibility in the excitation pattern.