Search Results (32)

Femtosecond laser two-photon polymerization (TPP) technology, known for its high precision and its ability to fabricate arbitrary 3D structures, has been widely applied in the production of various micro/nano optical devices, achieving significant advancements, particularly in the field of photonic wire bonding (PWB) for optical interconnects. Currently, research on optimizing both the optical loss and production reliability of polymeric photonic wires is still in its early stages. One of the key challenges is that inadequate metrology methods cannot meet the demand for multiphysical measurements in practical scenarios. This study utilizes novel in situ scanning electron microscopy (SEM) to monitor the working PWBs fabricated by TPP technology at the microscale. Optical and mechanical measurements are made simultaneously to evaluate the production qualities and to study the multiphysical coupling effects of PWBs. The results reveal that photonic wires with larger local curvature radii are more prone to plastic failure, while those with smaller local curvature radii recover elastically. Furthermore, larger cross-sectional dimensions contribute dominantly to the improved mechanical robustness. The optical-loss deterioration of the elastically deformed photonic wire is only temporary, and can be fully recovered when the load is removed. After further optimization based on the results of multiphysical metrology, the PWBs fabricated in this work achieve a minimum insertion loss of 0.6 dB. In this study, the multiphysical analysis of PWBs carried out by in situ SEM metrology offers a novel perspective for optimizing the design and performance of microscale polymeric waveguides, which could potentially promote the mass production reliability of TPP technology in the field of chip-level optical interconnection. Full article

This paper presents the design and fabrication of new types of polymer-based cantilevers for atomic force microscopy. The design and fabrication are aimed at the capability of 3D laser microprinting technology based on two-photon polymerization on a standard silicon substrate. IP-Dip commercial material from the Nanoscribe company was used for the fabrication of the designed cantilevers. The fabricated microprinted cantilevers facilitate precise manipulation at the nanoscopic scale, which is essential for studying nanomaterials’ mechanical, electrical, and optical properties. The cantilevers’ flexibility allows for the integration of functional elements such as piezoelectric layers and optical fibers, enabling combined measurements of multiple physical parameters. Various cantilever geometries, including rectangular and V-shaped, are examined, and their resonance frequencies are calculated. The experimental process involves preparing the cantilevers on a silicon substrate and coating them with aluminum for enhanced reflectivity and conductivity. Scanning electron microscope analysis documents the precise form of prepared polymer cantilevers. The functionality of the probes is validated by scanning a step-height standard grating. This study demonstrates the versatility and precision of the fabricated cantilevers, showcasing their potential for large-area scans, living cell investigation, and diverse nanotechnology applications. Full article

Nonlinear microscopy (NM) enables us to investigate the morphology or monitor the physiological processes of the skin through the use of ultrafast lasers. Fiber (or fiber-coupled) lasers are of great interest because they can easily be combined with a handheld, scanning nonlinear microscope. This latter feature greatly increases the utility of NM for pre-clinical applications and in vivo tissue imaging. Here, we present a fiber-coupled, sub-ps Ti–sapphire laser system being optimized for in vivo, stain-free, 3D imaging of skin alterations with a low thermal load of the skin. The laser is pumped by a low-cost, 2.1 W, 532 nm pump laser and delivers 0.5–1 ps, high-peak-power pulses at a ~20 MHz repetition rate. The spectral bandwidth of the laser is below 2 nm, which results in a low sensitivity for dispersion during fiber delivery. The reduction in the peak intensity due to the increased pulse duration is compensated by the lower repetition rate of our laser. In our proof-of-concept imaging experiments, a ~1.8 m long, commercial hollow-core photonic bandgap fiber was used for fiber delivery. Fresh and frozen skin biopsies of different skin alterations (e.g., adult hemangioma, basal cell cancer) and an unaffected control were used for high-quality, two-photon excitation fluorescence microscopy (2PEF) and second-harmonic generation (SHG) z-stack (3D) imaging. Full article

Nonlinear optical (NLO) imaging has emerged as a promising plant cell imaging technique due to its large optical penetration, inherent 3D spatial resolution, and reduced photodamage; exogenous nanoprobes are usually needed for nonsignal target cell analysis. Here, we report in vivo, simultaneous 3D labeling and imaging of potato cell structures using plasmonic nanoprobe-assisted multimodal NLO microscopy. Experimental results show that the complete cell structure can be imaged via the combination of second-harmonic generation (SHG) and two-photon luminescence (TPL) when noble metal silver or gold ions are added. In contrast, without the noble metal ion solution, no NLO signals from the cell wall were acquired. The mechanism can be attributed to noble metal nanoprobes with strong nonlinear optical responses formed along the cell walls via a femtosecond laser scan. During the SHG-TPL imaging process, noble metal ions that crossed the cell wall were rapidly reduced to plasmonic nanoparticles with the fs laser and selectively anchored onto both sides of the cell wall, thereby leading to simultaneous 3D labeling and imaging of the potato cells. Compared with the traditional labeling technique that needs in vitro nanoprobe fabrication and cell labeling, our approach allows for one-step, in vivo labeling of plant cells, thus providing a rapid, cost-effective method for cellular structure construction and imaging. Full article

Information on the penetration depth, pathways, metabolization, storage of vehicles, active pharmaceutical ingredients (APIs), and functional cosmetic ingredients (FCIs) of topically applied formulations or contaminants (substances) in skin is of great importance for understanding their interaction with skin targets, treatment efficacy, and risk assessment—a challenging task in dermatology, cosmetology, and pharmacy. Non-invasive methods for the qualitative and quantitative visualization of substances in skin in vivo are favored and limited to optical imaging and spectroscopic methods such as fluorescence/reflectance confocal laser scanning microscopy (CLSM); two-photon tomography (2PT) combined with autofluorescence (2PT-AF), fluorescence lifetime imaging (2PT-FLIM), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and reflectance confocal microscopy (2PT-RCM); three-photon tomography (3PT); confocal Raman micro-spectroscopy (CRM); surface-enhanced Raman scattering (SERS) micro-spectroscopy; stimulated Raman scattering (SRS) microscopy; and optical coherence tomography (OCT). This review summarizes the state of the art in the use of the CLSM, 2PT, 3PT, CRM, SERS, SRS, and OCT optical methods to study skin penetration in vivo non-invasively (302 references). The advantages, limitations, possibilities, and prospects of the reviewed optical methods are comprehensively discussed. The ex vivo studies discussed are potentially translatable into in vivo measurements. The requirements for the optical properties of substances to determine their penetration into skin by certain methods are highlighted. Full article

The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy. Full article

The Neurovascular Unit (NVU), composed of glia (astrocytes, oligodendrocytes, microglia), neurons, pericytes and endothelial cells, is a dynamic interface ensuring the physiological functioning of the central nervous system (CNS), which gets affected and contributes to the pathology of several neurodegenerative diseases. Neuroinflammation is a common feature of neurodegenerative diseases and is primarily related to the activation state of perivascular microglia and astrocytes, which constitute two of its major cellular components. Our studies focus on monitoring in real time the morphological changes of perivascular astrocytes and microglia, as well as their dynamic interactions with the brain vasculature, under physiological conditions and following systemic neuroinflammation triggering both microgliosis and astrogliosis. To this end, we performed 2-photon laser scanning microscopy (2P-LSM) for intravital imaging of the cortex of transgenic mice visualizing the dynamics of microglia and astroglia following neuroinflammation induced by systemic administration of the endotoxin lipopolysaccharide (LPS). Our results indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, an event that most possibly contributes to a loss of blood–brain barrier (BBB) integrity. At the same time, microglial cells become activated and exhibit a higher extent of physical contact with the blood vessels. These dynamic responses of perivascular astrocytes and microglia are peaking at 4 days following LPS administration; however, they still persist at a lower level at 8 days after LPS injection, revealing incomplete reversal of inflammation affecting the glial properties and interactions within the NVU. Full article

The mechanical properties of two-photon-polymerised (2PP) polymers are highly dependent on the employed printing parameters. In particular, the mechanical features of elastomeric polymers, such as IP-PDMS, are important for cell culture studies as they can influence cell mechanobiological responses. Herein, we employed optical-interferometer-based nanoindentation to characterise two-photon-polymerised structures manufactured with varying laser powers, scan speeds, slicing distances, and hatching distances. The minimum reported effective Young’s modulus (YM) was 350 kPa, while the maximum one was 17.8 MPa. In addition, we showed that, on average, immersion in water lowered the YM by 5.4%, a very important point as in the context of cell biology applications, the material must be employed within an aqueous environment. We also developed a printing strategy and performed a scanning electron microscopy morphological characterisation to find the smallest achievable feature size and the maximum length of a double-clamped freestanding beam. The maximum reported length of a printed beam was 70 µm with a minimum width of 1.46 ± 0.11 µm and a thickness of 4.49 ± 0.05 µm. The minimum beam width of 1.03 ± 0.02 µm was achieved for a beam length of 50 µm with a height of 3.00 ± 0.06 µm. In conclusion, the reported investigation of micron-scale two-photon-polymerized 3D IP-PDMS structures featuring tuneable mechanical properties paves the way for the use of this material in several cell biology applications, ranging from fundamental mechanobiology to in vitro disease modelling to tissue engineering. Full article

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720. Full article

Upconversion materials have numerous potential applications in light energy utilization due to their unique optical properties. The use of visible light excitation to obtain ultraviolet emission is a promising technology with broad application prospects, while relevant research is absent. A series of Pr³⁺, Gd³⁺ doped Y₇O₆F₉ phosphors were synthesized by traditional solid–state reaction. X-ray diffraction, scanning electronic microscopy, steady-state photoluminescence spectra, a decay dynamic, and upconversion emission spectra of the samples were studied. Under the excitation of 238 nm, the energy transfer from Pr³⁺ to Gd³⁺ was realized and a strong ultraviolet B emission due to the ⁶P_7/2→⁸S_7/2 transition of the Gd³⁺ ions was achieved. Under the excitation of a 450 nm blue laser, Pr³⁺ absorbed two blue photons to realize the upconversion process and then transferred the energy to Gd³⁺ to obtain the ultraviolet B emission. Full article

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca²⁺ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca²⁺ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca²⁺ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies. Full article

Three dimensional (3D) perovskite solar cells (PSCs) are a promising candidate for third-generation photovoltaics (PV) technology, which aims to produce efficient photon conversion devices to electricity using low-cost fabrication processes. Hybrid organic-inorganic perovskites for-lmed using low-cost solution processing are explored here, which have experienced a stupendous rise in power conversion efficiency (PCE) over the past decade and serve as a prime candidate for third-generation PV systems. While significant progress has been made, the inherent hygroscopic nature and stability issue of the 3D perovskites (3DPs) are an impediment to its commercialization. In this work, we have studied two-dimensional (2D) organometallic halide (CH₃(CH₂)₃NH₃)₂(CH₃NH₃)_n−1Pb_nI_3n+1) layered perovskites in the Ruddlesden Popper structure, represented as BA₂MA₃Pb₄I₁₃ for the n = 4 formulation, for both photoabsorbers in a two-terminal architecture and solar cells, given that these material are considered to be inherently more stable. In the two-terminal photo absorber devices, the photocurrent and responsivity were measured as a function of incoming laser wavelength, where the location of the peak current was correlated to the emission spectrum arising from the 2DP film using photoluminescence (PL) spectroscopy. The 2D (BA)2(MA)3Pb4I13 films were then integrated into an n-i-p solar cell architecture, and PV device figures of merit tabulated, while our 3D MAPbI₃ served as the reference absorber material. A comparative study of the 3DP and 2DP film stability was also conducted, where freshly synthesized films were inspected on FTO substrates and compared to those exposed to elevated humidity levels, and material stability was gauged using various material characterization probes, such as PL and UV-Vis optical absorption spectroscopy, scanning electron microscopy and X-ray diffraction. While the PCE of the 3D-PSCs was higher than the 2D-PSCs, our results confirm the enhanced environmental stability of the 2DP absorber films compared to the 3DP absorbers, suggesting their promise to address the stability issue broadly encountered in 3D PSCs toward third-generation PV technology. Full article

The stratum corneum (SC) forms a strong barrier against topical drug delivery. Therefore, understanding the penetration depth and pathways into the SC is important for the efficiency of drug delivery and cosmetic safety. In this study, TPT-FLIM (two-photon tomography combined with fluorescence lifetime imaging) was applied as a non-invasive optical method for the visualization of skin structure and components to study penetration depths of exemplary substances, like hydrophilic propylene glycol (PG), sodium fluorescein (NaFl) and lipophilic Nile red (NR) into porcine ear skin ex vivo. Non-fluorescent PG was detected indirectly based on the pH-dependent increase in the fluorescence lifetime of SC components. The pH similarity between PG and viable epidermis limited the detection of PG. NaFl reached the viable epidermis, which was also proved by laser scanning microscopy. Tape stripping and confocal Raman micro-spectroscopy were performed additionally to study NaFl, which revealed penetration depths of ≈5 and ≈8 μm, respectively. Lastly, NR did not permeate the SC. We concluded that the amplitude-weighted mean fluorescence lifetime is the most appropriate FLIM parameter to build up penetration profiles. This work is anticipated to provide a non-invasive TPT-FLIM method for studying the penetration of topically applied drugs and cosmetics into the skin. Full article

The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases. Full article

Lignin is one of the most studied and analyzed materials due to its importance in cell structure and in lignocellulosic biomass. Because lignin exhibits autofluorescence, methods have been developed that allow it to be analyzed and characterized directly in plant tissue and in samples of lignocellulose fibers. Compared to destructive and costly analytical techniques, fluorescence microscopy presents suitable alternatives for the analysis of lignin autofluorescence. Therefore, this review article analyzes the different methods that exist and that have focused specifically on the study of lignin because with the revised methods, lignin is characterized efficiently and in a short time. The existing qualitative methods are Epifluorescence and Confocal Laser Scanning Microscopy; however, other semi-qualitative methods have been developed that allow fluorescence measurements and to quantify the differences in the structural composition of lignin. The methods are fluorescence lifetime spectroscopy, two-photon microscopy, Föster resonance energy transfer, fluorescence recovery after photobleaching, total internal reflection fluorescence, and stimulated emission depletion. With these methods, it is possible to analyze the transport and polymerization of lignin monomers, distribution of lignin of the syringyl or guaiacyl type in the tissues of various plant species, and changes in the degradation of wood by pulping and biopulping treatments as well as identify the purity of cellulose nanofibers though lignocellulosic biomass. Full article