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The accurate retrieval of Solar-Induced chlorophyll Fluorescence (SIF) is a pivotal target for Earth Observation since SIF can be easily monitored through optical remote sensing and provides unique information concerning the vegetation health status. Here, we propose i-φ-MaLe (metti il nome per esteso), a novel algorithm, which couples the Fourier analysis with a supervised machine learning-based procedure trained with the atmosphere-canopy radiative transfer (RT) SCOPE model. i-φ-MaLe is the first method able to simultaneously retrieve, from the vegetation reflectance spectra, the Top Of Canopy SIF spectrum, the SIF spectrum corrected for leaf/canopy reabsorption (i.e. at photosystem level), the quantum efficiency (Fqe) and three canopy-related biophysical parameters (Leaf Area Index - LAI, Chlorophyll content - Cab and APAR) in few milliseconds. Validation procedures, based on the analysis of RT simulations, demonstrated that i-φ-MaLe, in experimental conditions (signal to noise ratio – SNR >= 500), estimates each biophysical parameter and SIF spectrum with a relative root mean square error (RRMSE) lower than 5%. In order to investigate the seasonal and daily dynamics of SIF, LAI, Cab, Fqe and APAR, the method has been also applied to field experimental data collected in the context of the AtmoFLEX and FLEXSense ESA campaigns, both at top-of-canopy (TOC) and tower (~100 meters) levels. Concerning the TOC scenario, the retrieved annual dynamic for SIF spectra has been compared with the results obtained by inversion-based methods, showing a good consistency amongthe different approaches (RRMSE ~ 10%). Moreover, SIF daily and annual dynamics, retrieved by excluding the oxygen spectral bands affected by the atmospheric reabsorption, have been investigated for high tower measurements. . In this context, i-φ-MaLe provided promising results that can integrate and possibly overcome complex and computationally expensive atmospheric compensation techniques actually needed to retrieve fluorescence from oxygen absorptions bands. This study demonstrates a promising potential to exploit ground and tower spectral measurements with advanced processing algorithms, for improving our understanding on the link between canopy structure and physiological functioning of plants. Moreover, i-φ-MaLe can be straightforwardly employed to process reflectance spectra and open new perspectives in fluorescence retrieval at different scales.

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. The elastic properties of the microstructures with different (circular and square) cross-sections are tested by Atomic Force Microscopy and by studying the deflection-flow dependence in microfluidic experiments at intermediate Reynolds numbers (Rey ≲ 150). The retrieved Young's modulus of the fabricated matrix (100 kPa ≤ E ≤ 4 MPa) falls in the range of most typical biological tissues and solely depends on the laser fabrication intensity. The elastic constant of the microstructures falls in the range of 0.8 nN μm−1 ≤ k ≤ 50 nN μm−1, and fully agrees with the macroscopic Euler Bernoulli theory. For soft microstructures (0.8 nN μm−1 ≤ k ≤ 8 nN μm−1) we reveal undamped bending oscillations under continuous microfluidic flow, corresponding to ∼10% of the total structure deflection. This behavior is ascribed to the coupling of the viscoelasticity and non-linear elasticity of the polymer matrix with non-linear dynamics arising from the time-dependent friction coefficient of the bendable microstructures. We envision that similar instabilities may lead to the development of promising energy conversion nanoplatforms.

The Ras/cAMP/PKA pathway is one of the best studied signalling pathway in the budding yeast that regulates cellular responses to nutrients availability and stress. The cAMP levels and the PKA activity are also subjected to a strong negative feedback that operates either through the activity of the phosphodiesterase Pde1 and also on the amount of Ras2-GTP. We have previously made and simulated a dynamic model of the whole pathway and our results suggest the existence of stable oscillatory states that depend on the activity of the RasGEF (Cdc25) and RasGAP (Ira proteins) (Pescini et al. Biotechnol Adv 30, 99-107, 2012). Stochastic oscillations related to activity of the pathway were reported by looking at the nuclear localization of the trascription factors Msn2 and Msn4 (Gamedia-Torres et al. Curr Biol 17, 1044-9, 2007). In particular Medvedik et al. (PloS Biol 5, 2330-41, 2007) reported stable oscillations of the nuclear accumulation of Msn2 in condition of limited glucose availabiliy.We were able to reproduce the periodic accumulation of Msn2-GFP protein in yeast cells under condition of limiting glucose, and we tried to detect also in the same condition oscillations of cAMP levels in single yeast cells. We used a specific Fluorescence Resonance Energy Transfer (FRET) sensor based on a fusion protein between CFP-EPAC-YFP expressed in yeast cells. The FRET between CFP and YFP is controlled by cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in single yeast cell for a relative long time and a peak of cAMP was normally detected after addition of glucose to derepressed cells (Colombo et al. Biochem Biophys Res Commun 487, 594-99, 2017). Using this method we were able to detect noisy oscillations of cAMP levels in single yeast cells under condition of nutritional stress caused by limiting glucose availability (0.1%). We used Spectral analysis to discriminate between true oscillations and random noise. The oscillations were characterized by period of about 4-5 min, close to that observed for Msn2-GFP oscillations.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Prussian blue (PB) is a coordination polymer based on the Fe2+…C[tbnd]N…Fe3+ sequence. It is an FDA-approved drug, intended for oral use at the acidic pH of the stomach and of most of the intestine track. However, based on FDA approval, a huge number of papers proposed the use of PB nanoparticles (PBnp) under “physiological conditions”, meaning pH buffered at 7.4 and high saline concentration. While most of these papers report that PBnp are stable at this pH, a small number of papers describes instead PBnp degradation at the same or similar pH values, i.e. in the 7–8 range. Here we give a definitively clear picture: PBnp are intrinsically unstable at pH ≥ 7, degrading with the fast disappearance of their 700 nm absorption band, due to the formation of OH- complexes from the labile Fe3+ centers. However, we show also that the presence of a polymeric coating (PVP) can protect PBnp at pH 7.4 for over 24 h. Moreover, we demonstrate that when “physiological conditions” include serum, a protein corona is rapidly formed on PBnp, efficiently avoiding degradation. We also show that the viability of PBnp-treated EA.hy926, NCI-H1299, and A549 cells is not affected in a wide range of conditions that either prevent or promote PBnp degradation.

Heating has been recently used as an alternative application to electrical stimulation to modulate excitability and to induce neuritogenesis and the expression of neuronal markers, but a long-term functional differentiation has not been described so far. Here we present the results obtained by a new approach for scalable thermal stimulation on the behavior of a model of dorsal root ganglion neurons, the F-11 cell line. Initially, we performed experiments of bulk stimulation in incubator for different time intervals and temperatures, and significant differences in neurite elongation and in electrophysiological properties were observed in cultures exposed at 41,5°C for 30 minutes. Thus, we exposed the cultures to the same temperature increase by irradiating, with a near infrared laser, a disc of Prussian Blue nanoparticles and poly-vinyl alcohol, that we stuck on the outer surface of the petri dish. In irradiated cells neurites were significantly longer and the electrophysiological properties (action potential firing frequency and spontaneous activity) were significantly increased compared to the control. These results suggest that a targeted thermal stimulation could be a promising technique to induce differentiation and support the future application of this method as a strategy to modify neuronal behavior in vivo.

Bacterial contamination is a severe issue that affects medical devices, hospital tools and surfaces. When microorganisms adhere to a surface (e.g., medical devices or implants) they can develop into a biofilm, thereby becoming more resistant to conventional biocides and disinfectants. Nanoparticles can be used as an antibacterial agent in medical instruments or as a protective coating in implantable devices. In particular, attention is being drawn to photothermally active nanoparticles that are capable of converting absorbed light into heat. These nanoparticles can efficiently eradicate bacteria and biofilms upon light activation (predominantly near the infrared to near-infrared spectral region) due a rapid and pronounced local temperature increase. By using this approach new, protective, antibacterial surfaces and materials can be developed that can be remotely activated on demand. In this review, we summarize the state-of-the art regarding the application of various photothermally active nanoparticles and their corresponding nanocomposites for the light-triggered eradication of bacteria and biofilms.

The current protocols for biocompatibility assessment of biomaterials, based on histopathology, require the sacrifice of a huge number of laboratory animals with an unsustainable ethical burden and remarkable cost. Intravital microscopy techniques can be used to study implantation outcomes in real time though with limited capabilities of quantification in longitudinal studies, mainly restricted by the light penetration and the spatial resolution in deep tissues. We present the outline and first tests of a novel chip which aims to enable longitudinal studies of the reaction to the biomaterial implant. The chip is composed of a regular reference microstructure fabricated via two-photon polymerization in the SZ2080 resist. The geometrical design and the planar raster spacing largely determine the mechanical and spectroscopic features of the microstructures. The development, in-vitro characterization and in vivo validation of the Microatlas is performed in living chicken embryos by fluorescence microscopy 3 and 4 days after the implant; the quantification of cell infiltration inside the Microatlas demonstrates its potential as novel scaffold for tissue regeneration.

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. We start from camera-based temperature detection via Stefan–Boltzmann’s law, and elucidate the interplay of the camera point-spread-function and pixelated sensor size with the excitation beam waist in defining the amplitude of the measured temperature variations. This can be accomplished by the numerical solution of the three-dimensional heat equation in the presence of modulated laser illumination on the sample, which is characterized in terms of thermal diffusivity, conductivity, thickness, and concentration of photo-thermal species. We apply our data-analysis protocol to murine B16 melanoma biopsies, where melanin is mapped and quantified in label-free configuration at sub-diffraction 40 µm resolution. Our results, validated by an unsupervised machine-learning analysis of hematoxylin-and-eosin images of the same sections, suggest potential impact of super-resolved thermography in complementing standard histopathological analyses of melanocytic lesions.

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. By exploiting the photo-thermal effect primed by melanin absorption of visible laser light we obtain label-free super-resolution far-infrared thermal images of tissue sections where melanin is spatially mapped at sub-diffraction 40-μm resolution. Based on the finite-element simulation of the full 3D heat transfer model, we are able to convert temperature maps into quantitative images of the melanin molar concentration on B16 murine melanoma biopsies, with 4·10-4 M concentration sensitivity. Being readily applicable to human melanoma biopsies in combination with hematoxylin-and-eosin staining, the proposed approach could complement traditional histopathology in the characterization of pigmented lesions ex-vivo.

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. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. Less often, the two approaches have been combined and active nanomaterials have been embedded in the microstructures. The aim of this review is to give a short overview of the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photothermal activity, chemical swelling and chemical sensing.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca(2+) mobilization. In DCs, Ca(2+) mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP(3)). Here, we showed that the IP(3) receptor 3 (IP(3)R3) and ITPKB, a kinase that converts IP(3) to inositol 1,3,4,5-tetrakisphosphate (IP(4)), were both necessary for Ca(2+) mobilization and NFAT activation in mouse and human DCs. A pool of IP(3)R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca(2+) mobilization through plasma membrane-localized IP(3)R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

In the context of nosocomial infections, there is an urgent need to develop efficient nanomaterials (NMs) with antibacterial properties for the prevention of infection diseases. Metal oxide nanoparticles (MeO-NPs) are promising candidates for the development of new antibacterial textiles. However, the direct exposure to MeO-NPs and MeO-coated NMs through skin contact could constitute a severe hazard for human health. In this work, the toxicity of copper and zinc oxide (CuO, ZnO) NPs antimicrobial-coated textiles was assessed on an in vitro reconstructed 3D model of epidermis. Thus, MeO-NPs and extracts from MeO-coated NMs were tested on EpiDerm™ skin model according to OECD TG 431 (Corrosion Test) and 439 (Irritation Test), respectively. Skin surface fluids composition is a crucial aspect to be considered in the development of NMs that have to encounter this tissue. So, for the irritation test, coated textiles were extracted in artificial sweat solutions at pH 4.7 and 6.5. Skin tissue viability, pro-inflammatory interleukin-8 secretion and morphological alteration of intermediate and actin filaments of keratinocytes were evaluated after 18 h exposure to extracts from CuO- and ZnO-coated textiles. Analysis of extracts at the two pH conditions indicated that released ions and not NPs are involved in promoting adverse effects on epidermis. Since Cu2+ and Zn2+ ions are known to penetrate epidermis, Balb/3 T3 cells were used as model of dermis. Fibroblasts viability was investigated after the exposure to trans-epidermis permeated ions, collected from EpiDerm™ basal supernatants, and to extracts, as representative of a direct interaction of ions with dermis cells by wounded skin. From our data we can conclude that: 1) skin surface fluids composition is a key parameter for the stability of NPs-coated textiles; 2) MeO ions released from coated textiles can deeply affect the epidermal tissue and the underlying dermal cells upon trans-epidermal permeation; 3) skin barrier integrity is a fundamental prerequisite that should be taken into account during the assessment of NMs safety by direct contact exposure.

This work presents the design and experimental validation of a photoacoustic setup that emulates the acoustic signal generated by a 20 MeV proton beam by ionoacoustic effect. This system serves as a practical tool to experimentally test ionoacoustic detectors in a simple yet representative setup prior to performing beam tests at accelerator facilities. To emulate the ionoacoustic effect the hereby presented Proton Sound Detector Testbench (ProSD-TB) uses a 70 W peak power pulsed laser and a specifically designed target absorber. The experimental validation of the ProSD-TB shows that it is capable to emulate a 20 MeV proton beam ionoacoustic signal in terms of signal amplitude (few Pa at the sensor surface) and frequency (2 MHz). The time domain signal generated by the ProSD-TB is acquired by means of a 64-ch Proton Sound Detector composed by a multichannel acoustic sensor, a dedicated analog front-end and signal processing. The ProSD-TB is validated against a 20 MeV ionoacoustic signal both in time domain and by comparing the 20 MeV dose deposition profile to an acoustic image generated by the Proton Sound Detector.

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. Under NIR light irradiation with low intensities (0.35 W/cm2) these films demonstrated a pronounced photo-thermal effect: &Delta, Tmax up to 26.4 &deg, C for the GNS-containing films and &Delta, Tmax up to 45.8 &deg, C for the PB-containing films. In the latter case, such a local temperature increase demonstrated a remarkable effect on a Gram-negative strain (P. aeruginosa) killing (84% of dead bacteria), and a promising effect on a Gram-positive strain (S. aureus) eradication (69% of dead bacteria). The fabricated films are promising prototypes for further development of lightweight surfaces with efficient antibacterial action that can be remotely activated on demand.

Brand new biomaterials, intended to be used on humans, must undergo in vivo quantification standardized, expensive and unethical procedures mainly based on histopathological analysis, from dissections, as defined by the ISO 10993 normative set. The aim is to prove the biomaterials biocompatibility. There exist no methods based on intravital microscopy able to satisfy the normative quantification requirements both reducing the number of employed animals and related costs. We developed a miniaturized imaging window, the Microatlas, which allows subcutaneous repeated observations in vivo of the foreign body reactions, for example to the implantation of a biomaterial. Confocal and twophoton microscopy inspections at Microatlas implantation sites demonstrated growth of the recipient tissue inside the microgrids both with micro vascularization formation and collagen generation. In conclusion, the Microatlas guided in vivo a quantifiable localized reaction inside its microscaffold, both in terms of cell repopulation, collagen and capillary formation as a probable foreign body reaction.

Inkjet-printing of metal nanoparticles is a particularly promising technique for the fabrication and modification of surfaces with a multifunctional nature. Recently copper sulfide nanoparticles (CuS NPs) have attracted wide interest due to a range of valuable properties including long term stability, photo-thermal activity, ease of synthesis and low cost. In the present study, printed CuS patterns were successfully fabricated on latex coated paper substrates and characterized by means of atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), UV–Vis-NIR spectroscopy, and grazing incidence X-ray diffraction (GID). The resulted patterns displayed pronounced photo-thermal effect under Near Infrared Irradiation (NIR) even with relatively low laser power. Finally, by utilizing an automated real-time imaging platform it was possible to verify that the CuS printed film was not cytotoxic to human dermal fibroblast cells (HDF). The pronounced photo-thermal properties and nontoxic nature of these printed low-cost flexible CuS films make them promising candidates for fabrication of devices with localized photo-thermal effect suitable for biomedical applications.

Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (μMAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. μMAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply μMAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors ex-vivo and in-vivo. We develop a clustering algorithm to automatically group pixels with similar microstructural features. μMAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.

Thermal conductivity measurements play a crucial role in most areas of the applied physical sciences, thereby constantly demanding for new non-invasive methods capable of providing high-resolution spatial mapping of absolute thermal conductivities on heterogeneous samples ranging from solid-state bulk materials to low-dimensionality structures. In this work, we lay the theoretical foundations and provide the experimental demonstration of a novel method for quantitative thermal conductivity mapping at tunable ∼10-μm resolution, by non-contact infrared photo-activated thermography. Starting from Fourier’s heat transfer law, we surmise a universal dependence of the thermal response of a laser-irradiated sample on its thermal conductivity, irrespectively of density and specific heat capacity. We demonstrate such a dependence over the three conductivity decades 0.1–100 W/mK by finite-element numerical simulations, and exploit it for proof-of-principle single-point thermal conductivity measurements on both thermally thick and thermally thin reference solid samples. We exemplify the feasibility of space-resolved measurements on eighteenth-century tin organ pipe fragments, where the product of the thermal conductivity times the sample thickness, imaged at 40-μm sub-diffraction resolution, is pointed out as a relevant parameter for the non-destructive characterization of the sample conservation state. By coupling temperature maps with the extraction of thermal properties at high spatial resolution, our approach significantly expands the capability of state-of-the-art infrared imaging technology in fully capturing the compositional heterogeneity and/or functional state of the imaged materials.

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. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour).

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. The collagen fibrils microscopic parameters (orientation and anisotropy) are analyzed at a mesoscopic level by quantifying their local spatial heterogeneity in histopathology sections (few mm in size) from two cancer xenografts in mice, in order to maximally discriminate different collagen organizations, allowing in this case to identify the tumor area with respect to the surrounding skin tissue. We show that the “fibril entropy” parameter, which describes the tissue order on a selected spatial scale, is the most effective in enlightening the tumor edges, opening the possibility of their automatic segmentation. Our method, therefore, combined with tissue morphology information, has the potential to become a support to standard histopathology in diseases diagnosis.

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. However, there is still a limitation on the signal-background due to the wave front distortion. We develop a diffractive optical microscope based on a single spatial light modulator (SLM) with no movable parts. We combine the correction of wave front distortions to the cross-correlation analysis of the flow dynamics. We use the SLM to shine arbitrary patterns of spots on the sample, to correct their optical aberrations, to shift the aberration corrected spot array on the sample for the collection of fluorescence images, and to measure flow velocities from the cross-correlation functions computed between couples of spots. The setup and the algorithms are tested on various microfluidic devices. By applying the adaptive optics correction algorithm, it is possible to increase up to 5 times the signal-to-background ratio and to reduce approximately of the same ratio the uncertainty of the flow speed measurement. By working on grids of spots, we can correct different aberrations in different portions of the field of view, a feature that allows for anisoplanatic aberrations correction. Finally, being more efficient in the excitation, we increase the accuracy of the speed measurement by employing a larger number of spots in the grid despite the fact that the two-photon excitation efficiency scales as the fourth power of this number: we achieve a twofold decrease of the uncertainty and a threefold increase of the accuracy in the evaluation of the flow speed.

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. In this study polyvinyl alcohol (PVA) hydrogel films containing non-toxic and highly photo-thermally active Prussian blue (PB) nanoparticles were fabricated. The confocal microscopy studies indicated a uniform nanoparticle distribution and a low degree of aggregation. Upon near-infrared (NIR; 700 and 800 nm) light irradiation of PVA-PB films, the local temperature increases rapidly and reaches a plateau (up to ΔT ≅ 78 °C), within ≈6-10 s under relatively low laser intensities, I ≅ 0.3 W cm-2. The high and localized increase of temperature on the fabricated films resulted in an efficient antibacterial effect on Pseudomonas aeruginosa (P. aeruginosa) bacteria. In addition, the localized photo-thermal effect was also sufficient to substantially mitigate biofilms growth.

Active thermal imaging is a valuable tool for the nondestructive characterization of the morphological properties and the functional state of biological tissues and synthetic materials. However, state-of-the-art techniques do not typically combine the required high spatial resolution over extended fields of view with the quantification of temperature variations. Here, we demonstrate quantitative far-infrared photo-thermal imaging at sub-diffraction resolution over millimeter-sized fields of view. Our approach combines the sample absorption of modulated raster-scanned laser light with the automated localization of the laser-induced temperature variations imaged by a thermal camera. With temperature increments ∼0.5–5 °C, we achieve a six-time gain with respect to our 350-μm diffraction-limited resolution with proof-of-principle experiments on synthetic samples. We finally demonstrate the biological relevance of sub-diffraction thermal imaging by retrieving temperature-based super-resolution maps of the distribution of Prussian blue nanocubes across explanted murine skin biopsies., Thermal characterization of biological tissues can traditionally provide either large fields of view or high resolution. By the development of super-resolution infrared photo-thermal imaging, the authors reconstruct temperature-based maps of absorptive centers in millimeter-sized biological samples.

Natural killer (NK) cells are critical players against tumors. The outcome of anti‐tumor vaccination protocols depends on the efficiency of NK‐cell activation, and efforts are constantly made to manipulate them for immunotherapeutic approaches. Thus, a better understanding of NK‐cell activation dynamics is needed. NK‐cell interactions with accessory cells and trafficking between secondary lymphoid organs and tumoral tissues remain poorly characterized. Here, we show that upon triggering innate immunity with lipopolysaccharide (LPS), NK cells are transiently activated, leave the lymph node, and infiltrate the tumor, delaying its growth. Interestingly, NK cells are not actively recruited at the draining lymph node early after LPS administration, but continue their regular homeostatic turnover. Therefore, NK cells resident in the lymph node at the time of LPS administration become activated and exert anti‐tumor functions. NK‐cell activation correlates with the establishment of prolonged interactions with dendritic cells (DCs) in lymph nodes, as observed by two‐photon microscopy. Close DC and NK‐cell contacts are essential for the localized delivery of DC‐derived IL‐18 to NK cells, a strict requirement in NK‐cell activation.

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (μMAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. μMAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply μMAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors ex-vivo and in-vivo. We develop a clustering algorithm to automatically group pixels with similar microstructural features. μMAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.

The study of the dynamics of biological systems requires one to follow relaxation processes in time with micron-size spatial resolution. This need has led to the development of different fluorescence correlation techniques with high spatial resolution and a tremendous (from nanoseconds to seconds) temporal dynamic range. Spatiotemporal information can be obtained even on complex dynamic processes whose time evolution is not forecast by simple Brownian diffusion. Our discussion of the most recent applications of image correlation spectroscopy to the study of anomalous sub- or superdiffusion suggests that this field still requires the development of multidimensional image analyses based on analytical models or numerical simulations. We focus in particular on the framework of spatiotemporal image correlation spectroscopy and examine the critical steps in getting information on anomalous diffusive processes from the correlation maps. We point out how a dual space-time correlative analysis, in both the direct and the Fourier space, can provide quantitative information on superdiffusional processes when these are analyzed through an empirical model based on intermittent active dynamics. We believe that this dual space-time analysis, potentially amenable to mathematical treatment and to the exact fit of experimental data, could be extended to include the rich phenomenology of subdiffusive processes, thereby quantifying relevant parameters for the various motivating biological problems of interest.