The Perception of the Body Condition of Cats and Dogs by French Pet Owners and the Factors Influencing Underestimation.

Managing pet obesity relies heavily on the active involvement of owners; however, a key challenge arises from misperceptions about their own pet's body condition. Given evolving societal dynamics like the body positivity movement, understanding owners' perceptions is increasingly pivotal. To evaluate the differences in owners' perception, this study compared the use of verbal and visual body condition score scales versus the established nine-point body condition score system. The factors linked to underestimation were further specifically investigated. Owners of healthy adult dogs and cats attending vaccination consultations in Veterinary Hospitals in France between 2020 and 2022 were recruited. They were required to assess their pets' body condition initially using an oral description and then with the nine-point BCS visual scale. Their assessments were then compared with the BCS determined by veterinary health care personnel, considered the primary investigator. A total of 304 dogs and 270 cats were included in the study. It was observed that 27% of dog owners and 24% of cat owners underestimated their pets' body condition. Among dog and cat owners, factors associated with the underestimation of body condition were the pets' overweight status and having children. This discovery emphasizes the need for a holistic One Health approach that prioritizes the health and well-being of both humans and their pets. When it comes to pet owners evaluating their pets' body condition, underestimation proved to be the predominant misperception. Addressing this issue requires comprehensive education to empower owners to recognize and comprehend their pets' overweight status, a critical step for the overall well-being of companion animals.

Influence of photothermal and plasma-mediated nano-processes on fluence thresholds for ultrafast laser-induced cavitation around gold nanoparticles.

Laser fluence thresholds of ultrafast excitation of vapor bubbles around gold nanoparticles are determined experimentally. An optical scattering technique of limited minimum bubble size resolution is employed and analyzed for that purpose. Measurements were performed for spherical gold nanoparticles of varying sizes (40-200 nm) and for laser pulses of varying pulse width (55 fs to 4.3 ps) to estimate the limits where the evaluated thresholds are attributed to either plasma-mediated or photothermal cavitation. Furthermore, thresholds were obtained by double 55 fs pulsed excitation (varying delay 0.0-4.3 ps), providing insights into the dynamics of the excited plasma. A relationship is established between particle properties, (size, near-field amplification factor, and absorption efficiency) and the crossover pulse width of the transition from plasma-mediated to photothermal cavitation. Further, by comparing theory and experiments, we examine the approximative optical breakdown density of ∼10-21 cm-3 at a distance of 1-2 nm from the particle surface as a criterion of plasma-mediated cavitation around gold nanoparticles in analogy to the spinodal criterion for photothermal cavitation. For a given pulse width, the breakdown density appears to be nearly size-independent, establishing the aforesaid criterion applicable. However, a small pulse width dependence of the breakdown density is still observed. Based on these criteria, a comparison is further provided between theoretical thresholds of cavitation and the ones of detectable bubbles. An increasing discrepancy is observed between them with decreasing size for the case of photothermal cavitation. For plasma-mediated cavitation, the latter discrepancy is seemingly smaller, presumably due to the highly nonlinear nature of the process.

Single Pulse Nanosecond Laser-Stimulated Targeted Delivery of Anti-Cancer Drugs from Hybrid Lipid Nanoparticles Containing 5 nm Gold Nanoparticles.

Encapsulating chemotherapeutic drugs like doxorubicin (DOX) inside lipid nanoparticles (LNPs) can overcome their acute, systematic toxicity. However, a precise drug release at the tumor microenvironment for improving the maximum tolerated dose and reducing side effects has yet to be well-established by implementing a safe stimuli-responsive strategy. This study proposes an integrated nanoscale perforation to trigger DOX release from hybrid plasmonic multilamellar LNPs composed of 5 nm gold (Au) NPs clustered at the internal lamellae interfaces. To promote site-specific DOX release, a single pulse irradiation strategy is developed by taking advantage of the resonant interaction between nanosecond pulsed laser radiation (527 nm) and the plasmon mode of the hybrid nanocarriers. This approach enlarges the amount of DOX in the target cells up to 11-fold compared to conventional DOX-loaded LNPs, leading to significant cancer cell death. The simulation of the pulsed laser interactions of the hybrid nanocarriers suggests a release mechanism mediated by either explosive vaporization of thin water layers adjacent to AuNP clusters or thermo-mechanical decomposition of overheated lipid layers. This simulation indicates an intact DOX integrity following irradiation since the temperature distribution is highly localized around AuNP clusters and highlights a controlled light-triggered drug delivery system.

Sensitive detection of electric field-induced second harmonic signals.

We demonstrate sensitive electric field measurements by coherent homodyne amplification of the electric field induced second harmonic generation (E-FISH) technique. In the process of E-FISH, an applied electric field breaks the centrosymmetry of an otherwise homogeneous medium, in turn promoting the generation of the second harmonic frequency of an incident field. Due to weak third-order hyperpolarizability and the requirement of an applied field to break the symmetry, the E-FISH technique has been mainly used to study high fields, also requiring a strong optical field and sensitive detection. Here we superimpose the E-FISH signal with an auxiliary beam, also termed a local oscillator (LO), at double the incident frequency. Coherent superposition of the LO and the E-FISH output (LOE-FISH) allows for a homodyne amplification of the otherwise weak nonlinear signal. We have demonstrated an increase of signal-to-noise by a factor of seven, which results in a measurement time reduction of a factor of 49. This technique, LOE-FISH, has a number of advantages: detection with intensified detectors is not required. Furthermore, instead of millijoule pulsed lasers, we can work with microjoule pulsed lasers, which allows measuring at repetition rates of megahertz and opens single shot and real-time capability. The LOE-FISH technique increases in sensitivity at lower electric field values. Our work is a demonstration of the principle. Already with our first results from the demonstration, one can see the high potential of LOE-FISH.

Coiled-Coil-Based Biofunctionalization of 100 nm Gold Nanoparticles with the Trastuzumab Antibody for the Detection of HER2-Positive Cancer Cells.

We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.

Dynamic multispectral detection of bacteria with nanoplasmonic markers.

Culture-based diagnosis of bacterial diseases is a time-consuming technique that can lead not only to antibiotic resistance or bacterial mutation but also to fast-spreading diseases. Such mutations contribute to the fast deterioration of the patient's health and in some cases the death depending on the complexity of the infection. There is great interest in developing widely available molecular-level diagnostics that provide accurate and rapid diagnosis at the individual level and that do not require sophisticated analysis or expensive equipment. Here, we present a promising analytical approach to detect the presence of pathogenic bacteria based on their dynamic properties enhanced with nanoplasmonic biomarkers. These markers have shown greater photostability and biocompatibility compared to fluorescent markers and quantum dots, and serve as both a selective marker and an amplifying agent in optical biomedical detection. We show that a simple dark-field side- illumination technique can provide sufficiently high-contrast dynamic images of individual plasmonic nanoparticles attached to Escherichia coli (E. coli) for multiplex biodetection. Combined with numerical dynamic filtering, our proposed system shows great potential for the deployment of portable commercial devices for rapid diagnostic tests available to physicians in emergency departments, clinics and public hospitals as point-of-care devices.

Nonlinear thermal lensing of high repetition rate ultrafast laser light in plasmonic nano-colloids.

We report on experimental observations of phenomenological self-trapping in plasmonic colloids of varying plasmon peaks in the visible/near infrared. A femtosecond (fs) oscillator is used in both pulsed (35 fs, 76 MHz) and continuous wave (cw) operation for comparison. We show that for both modes and for all examined colloids (and under typically applied external focusing conditions in self-trapping studies in colloidal media) nonlinear propagation is governed by thermal defocusing of the focused beam, which precedes the steady-state regime reached by particle diffusion, even far from the plasmon resonance (or equivalently for non-plasmonic colloids, even for low absorption coefficients). A strategy for the utilization of high repetition fs pulses to mitigate thermal lensing and promote gradient force-induced self-trapping is discussed. Notably, nonlinear thermal lensing is further accompanied by natural convection due to the horizontal configuration of the setup. Under resonant illumination, for both fs and cw cases, we observe mode break-up of the beam profile, most likely due to azimuthal modulation instability. Importantly, time-resolved observations of the break-up indicate that in the fs case, thermal convection heat transfer is reduced in magnitude and significantly decoupled in time from thermal conduction, presumably due to temperature increase confinement near the particles. We anticipate that our findings will trigger interest toward the use of high repetition fs pulses for self-channeling applications in nano-colloids.

Porous Au-Ag Nanoparticles from Galvanic Replacement Applied as Single-Particle SERS Probe for Quantitative Monitoring.

Plasmonic nanostructures have raised the interest of biomedical applications of surface-enhanced Raman scattering (SERS). To improve the enhancement and produce sensitive SERS probes, porous Au-Ag alloy nanoparticles (NPs) are synthesized by dealloying Au-Ag alloy NP-precursors with Au or Ag core in aqueous colloidal environment through galvanic replacement reaction. The novel designed core-shell Au-Ag alloy NP-precursors facilitate controllable synthesis of porous nanostructure, and dealloying degree during the reaction has significant effect on structural and spectral properties of dealloyed porous NPs. Narrow-dispersed dealloyed NPs are obtained using NPs of Au/Ag ratio from 10/90 to 40/60 with Au and Ag core to produce solid core@porous shell and porous nanoshells, having rough surface, hollowness, and porosity around 30-60%. The clean nanostructure from colloidal synthesis exhibits a redshifted plasmon peak up to near-infrared region, and the large accessible surface induces highly localized surface plasmon resonance and generates robust SERS activity. Thus, the porous NPs produce intensely enhanced Raman signal up to 68-fold higher than 100 nm AuNP enhancement at single-particle level, and the estimated Raman enhancement around 7800, showing the potential for highly sensitive SERS probes. The single-particle SERS probes are effectively demonstrated in quantitative monitoring of anticancer drug Doxorubicin release.

Femtosecond nearly resonant self-focusing in gold nanorod colloids.

We evaluate the threshold power for self-focusing in gold nanorod colloids of varying concentration by a power limiting method in the femtosecond filamentation regime. The pulses are tuned near the longitudinal plasmon peak of the nanorods, leading to saturation of linear absorption and reshaping of the particles. We evaluated the last two effects by optical transmission measurements and spectroscopic analysis and estimated that considerable particle deformation does not occur before the collapse of the beam. We performed numerical simulations based on the experimental results, and evaluated only a subtle, monotonically increasing enhancement of the nonlinear refractive index of the host material (water) as the nanoparticles concentration increases. The role of higher-order contributions is discussed. Our work provides an alternative characterization approach of ultrafast nonlinearities in absorbing media. It further emphasizes that self-focusing of intense femtosecond pulses in gold nanocomposites is hampered by the ultrafast modulation of the susceptibility of the metal.

Multiplexed Plasmonic Nano-Labeling for Bioimaging of Cytological Stained Samples.

Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.

Optical power limiter in the femtosecond filamentation regime.

We present the use of a power limiting apparatus to evaluate ultrafast optical nonlinearities of transparent liquids (water and ethanol) in the femtosecond filamentation regime. The setup has been previously employed for the same purpose, however, in a longer pulsewidth (> 20 ps) regime, which leads to an ambiguous evaluation of the critical power for self-focusing. The uncertainty originates from the existence of a threshold power for optical breakdown well below the critical power for self-focusing within this timeframe. Contrarily, using the proposed apparatus in the femtosecond regime, we observe for the first time a unique optical response, which features the underlying physics of laser filamentation. Importantly, we demonstrate a dependence of the optical transmission of the power limiter on its geometrical, imaging characteristics and the conditions under which a distinct demarcation for the critical power for self-focusing can be determined. The result is supported by numerical simulations, which indicate that the features of the observed power-dependent optical response of the power limiting setup are physically related to the spontaneous transformation of the laser pulses into nonlinear conical waves.

Formation Pathways of Porous Alloy Nanoparticles through Selective Chemical and Electrochemical Etching.

Porous alloy nanomaterials are important for applications in catalysis, sensing, and actuation. Chemical and electrochemical etching are two methods to form porous nanostructures by dealloying bimetallic nanoparticles (NPs). However, it is not clear how the NPs evolve during these etching processes. Insight into the morphological and compositional transformations of the NPs during the etching is critical to understanding the nanoscale details of the dealloying process. Here, using in situ liquid phase transmission electron microscopy, the structural evolution of individual AuAg alloy NPs is tracked during both chemical and electrochemical etching of their Ag component. The observations show that the electrochemical etching produces NPs with more uniform pore sizes than the chemical etching and enables tuning the NPs porosity by modulating the electrochemical potential. The results show that at the initial stages of both etching methods, Au-rich passivation layer forms on the surface of the NPs, which is critical in preserving the NP's porous shell as pores form underneath this layer during the etching. These findings describing the selective etching and dealloying of AuAg NPs provide a critical insight needed to control the morphology and composition of porous multimetallic NPs, and paves the way for synthesizing nanomaterials with tailored chemical and physical properties for various applications.

Antibody-Functionalized Gold Nanostar-Mediated On-Resonance Picosecond Laser Optoporation for Targeted Delivery of RNA Therapeutics.

The rapid advances of genetic and genomic technology indicate promising therapeutic potential of genetic materials for regulating abnormal gene expressions causing diseases and disorders. However, targeted intracellular delivery of RNA therapeutics still remains a major challenge hindering the clinical translation. In this study, an elaborated plasmonic optoporation approach is proposed to efficiently and selectively transfect specific cells. The site-specific optoporation is obtained by tuning the spectral range of a supercontinuum pulsed picosecond laser in order for each individual cell binding gold nanostar with their unique resonance peak to magnify the local field strength in the near-infrared region and facilitate a selective delivery of small interfering RNA, messenger RNA, and Cas9-ribonucleoprotein into human retinal pigment epithelial cells. Numerical simulations indicate that optoporation is not due to a plasma-mediated process but rather due to a highly localized temperature rise both in time (few nanoseconds) and space (few nanometers). Taking advantage of the numerical simulation and fine-tuning of the optical strategy, the perforated lipid bilayer of targeted cells undergoes a membrane recovery process, important to retain their viability. The results signify the prospects of antibody functionalized nanostar-mediated optoporation as a simple and realistic gene delivery approach for future clinical practices.

OrgaNet: A Robust Network for Subcellular Organelles Classification in Fluorescence Microscopy Images.

Automatic identification of subcellular compartments of proteins in fluorescence microscopy images is an important task to quantitatively evaluate cellular processes. A common problem for the development of deep learning based classifiers is that there is only a limited number of labeled images available for training. To address this challenge, we propose a new approach for subcellular organelles classification combining an effective and efficient architecture based on a compact Convolutional Neural Network and deep embedded clustering algorithm. We validate our approach on a benchmark of HeLa cell microscopy images. The network both yields high accuracy that outperforms state of the art methods and has significantly small number of parameters. More interestingly, experimental results show that our method is strongly robust against limited labeled data for training, requiring four times less annotated data than usual while maintaining the high accuracy of 93.9%.

Single point single-cell nanoparticle mediated pulsed laser optoporation.

This article presents an optical platform for studying the dynamics of nanoparticle assisted pulsed laser optoporation of individual living cells. Here plasmonic nanoparticles (NPs) act as markers of the exact spatial position of living cell membranes and as an enhancer for localized pulsed laser perforation. High contrast NP imaging using reflected light microscopy (RLM) allows accurate and automatic laser targeting at individual NPs for spatially controlled laser optoporation of single cells at a single point. The NP imaging method is compatible with fluorescence microscopy and a cellular incubator that allows study of real-time perforation kinetics of live cells and the optomechanical interaction of NPs with membranes. These parameters are of great interest for the development and experimental implementation of the technology of pulsed laser optoporation and transfection applied to single living cells as well as to bulk-level assays.

Designable nanoplasmonic biomarkers for direct microscopy cytopathology diagnostics.

Direct microscopy interpretation of fine-needle biopsy cytological samples is routinely used by practicing cytopathologists. Adding possibility to identify selective and multiplexed biomarkers on the same samples and with the same microscopy technique can greatly improve diagnostic accuracy. In this article, we propose to use biomarkers based on designable plasmonic nanoparticles (NPs) with unique optical properties and excellent chemical stability that can satisfy the above-mentioned requirements. By finely controlling the size and composition of gold-silver alloy NPs and gold nanorods, the NPs plasmonic resonance properties, such as scattering efficiency and resonance peak spectral position, are adjusted in order to provide reliable identification and chromatic differentiation by conventional direct microscopy. Efficient darkfield NPs imaging is performed by using a novel circular side illumination adaptor that can be easily integrated into any microscopy setup while preserving standard cytopathology visualization method. The efficiency of the proposed technology for fast visual detection and differentiation of three spectrally distinct NP-markers is demonstrated in different working media, thus confirming the potential application in conventional cytology preparations. It is worth emphasizing that the presented technology does not interfere with standard visualization with immunohistochemical staining, but should rather be considered as a second imaging modality to confirm the diagnostics.

Laser-induced plasmon-mediated treatment of retinoblastoma in viscous vitreous phantom.

Retinoblastoma (RB) is a rare form of cancer of the retina most prevalent in young children. We successfully show that laser-induced cell disruption, mediated by gold plasmonic nanoparticle (NP), is a potential and efficient therapy to kill the cancerous cells. The proof of concept is demonstrated in vitro on cultured Y79 RB cancer cells with a nanosecond laser at 527 nm, for both attached cells at the bottom of a Petri dish and for floating, clustered cells in a viscous vitreous phantom comprised of hyaluronan. We report a cellular death of 82% after irradiation in classic culture medium and a cellular death of 98% in vitreous phantom, for similar number of NPs in each sample. It is found that the NPs efficiently penetrate the floating Y79 clusters cells in the vitreous phantom, leading to a cellular death of over 85% even within the centre of the aggregates. The proposed treatment technique is based on a similar nanosecond laser used to eliminate floaters in the vitreous, but with much lower (100-1000 times) fluences of 20 J cm-2 .

Cost-effective side-illumination darkfield nanoplasmonic marker microscopy.

We present the development of an innovative technology for quantitative multiplexed cytology analysis based on the application of spectrally distinctive plasmonic nanoparticles (NPs) as optical probes and on cost-effective side-illumination multispectral darkfield microscopy (SIM) as the differential NP imaging method. SIM is based on lateral illumination by arrays of discrete color RGB light emitting diodes (LEDs) of spectrally adjusted plasmonic NPs and consecutive detection by the conventional CMOS color camera. We demonstrate the enhanced contrast and higher resolution of our method for individual NP detection in the liquid medium and of NP markers attached on the cell membrane in a cytology preparation by comparing it to the conventional darkfield microscopy (DFM). The proposed illumination and detection system is compatible with current clinical microscopy equipment used by pathologists and can greatly simplify the adaptation of plasmonic NPs as novel reliable and stable biological multiplexed chromatic markers for biodetection and diagnosis.

A needle-like optofluidic probe enables targeted intracellular delivery by confining light-nanoparticle interaction on single cell.

Intracellular delivery of molecular cargo is the basis for a plethora of therapeutic applications, including gene therapy and cancer treatment. A very efficient method to perform intracellular delivery is the photo-activation of nanomaterials that have been previously directed to the cell vicinity and bear releasable molecular cargo. However, potential in vivo applications of this method are limited by our ability to deliver nanomaterials and light in tissue. Here, we demonstrate intracelullar delivery using a needle-like optofluidic probe capable of penetrating soft tissue. Firstly, we used the optofluidic probe to confine an intracellular delivery mixture, composed of 100 nm gold nanoparticles (AuNP) and membrane-impermeable calcein, in the vicinity of cancer cells. Secondly, we delivered nanosecond (ns) laser pulses (wavelength: 532 nm; duration: 5 ns) using the same probe and without introducing a AuNP cells incubation step. The AuNP photo-activation caused localized and reversible disruption of the cell membrane, enabling calcein delivery into the cytoplasm. We measured 67% intracellular delivery efficacy and showed that the optofluidic probe can be used to treat cells with single-cell precision. Finally, we demonstrated targeted delivery in tissue (mouse retinal explant) ex vivo. We expect that this method can enable nanomaterial-assisted intracellular delivery applications in soft tissue (e.g. brain, retina) of small animals.