Search Results (54)

We report a 1100 nm all-polarization-maintaining (all-PM) fiber laser based on gain-managed nonlinear amplification (GMNA), and demonstrate its capability for in vivo two-photon imaging. The home-built fiber oscillator functioned using a nonlinear amplification loop mirror (NALM), delivering a 38.1 MHz, 13.8 mW, 1024 nm signal laser. The pre-chirp management (PCM) was incorporated with GMNA to enable efficient nonlinear amplification. The system ultimately generated pulses with an energy of 110 nJ and a duration of 56 fs, with the corresponding 10 dB spectral range spanning from 1041 nm to 1117 nm. This 1100 nm ultrafast fiber laser provides a convenient light source for multi-photon microscopy (MPM). Full article

Self-assembled nanosphere structures are widely used as bottom-up platforms for ordered micro- and nanostructures, with applications in photonic crystals, sensing platforms, functional coatings, drug delivery, and nanosphere lithography. Their performance and reproducibility depend on structural order, packing density, interparticle spacing, and defect density and distribution. Thus, reliable imaging and quantitative defect detection are needed for quality evaluation and process optimization. This review provides an overview of defect characteristics, imaging systems, and defect detection algorithms for self-assembled nanosphere structures. It first introduces representative zero-, one-, two-, and three-dimensional assemblies, followed by a summary of common defects, including vacancies, interstitial particles, dislocations, grain boundaries, stacking faults, voids, and cracks. Optical microscopy, electron microscopy, atomic force microscopy, scanning near-field optical microscopy, and correlative techniques are compared in terms of resolution, field of view, temporal resolution, contrast mechanism, in situ capability, and compatibility with feedback control. Algorithmic approaches are also reviewed, encompassing classical image processing, machine learning, and deep learning, along with their applications in segmentation, localization, classification, and high-throughput analysis. Overall, reliable defect inspection requires integrated workflows. These workflows should combine appropriate imaging systems, image quality control, transferable algorithms, standardized datasets, and closed-loop feedback. Full article

Glucose-stimulated insulin secretion is the central functional readout of pancreatic islets, yet existing assays often require offline processing or pooling of multiple islets, limiting real-time assessment of single-islet function. Here we report a microscopy-compatible islet-on-a-chip (IOC) integrated with light scattering-based broadband backscattering confocal microscopy (BBCM) for continuous, label-free optical readout of insulin secretion dynamics in functional human islets. Fabricated using two-photon polymerization, the IOC-BBCM sensor stabilizes single human islets under continuous perfusion for high-resolution optical interrogation. The sensor identifies insulin-rich β-cells label-free, as confirmed by insulin immunostaining, and monitors granule depletion and redistribution during glucose and potassium chloride (KCl) stimulation, matching ELISA-quantified insulin secretion from the same perfused islets. This modular sensor provides a non-destructive, label-free approach for monitoring stimulus-linked secretion dynamics from individual human islets and should support longitudinal studies of islet function. Full article

Eleven novel asymmetric pyrimidine derivatives were synthesized. The pyrimidine core was functionalized with a coumarin chromophore and a pro-mesogenic fragment bearing either chiral or linear alkyl chains of variable length and substitution patterns. The thermal properties were investigated using polarized optical microscopy, differential scanning calorimetry, and small-angle X-ray scattering, revealing that only selected derivatives exhibited liquid crystalline phases with ordered columnar or smectic organizations. Linear and nonlinear optical properties were characterized by UV–Vis absorption, fluorescence spectroscopy, two-photon absorption, and second-harmonic generation. Optical responses were found to be highly sensitive to the substitution pattern: derivatives functionalized at the 4 and 3,4,5 positions exhibited enhanced 2PA cross-sections and pronounced SHG signals, whereas variations in alkyl chain length exerted only a minor influence. Notably, compounds forming highly ordered non-centrosymmetric mesophases produced robust SHG-active thin films. Importantly, strong SHG responses were obtained without the need for a chiral center, as the inherent asymmetry of the linear alkyl chain derivatives was sufficient to drive self-organization into non-centrosymmetric materials. These results demonstrate that asymmetric pyrimidine-based architectures combining π-conjugation and controlled supramolecular organization are promising candidates for nonlinear optical applications such as photonic devices, multiphoton imaging, and optical data storage. Full article

This study evaluates the efficacy of a novel moisturizing cream using a sodium lauryl sulfate (SLS)-induced skin damage model, supported by advanced imaging with two-photon microscopy (TPM). TPM’s capabilities allow for in-depth, non-invasive visualization of skin repair processes, surpassing traditional imaging methods. The innovative formulation of the cream includes ceramide NP, ceramide NS, ceramide AP, lactobacillus/soybean ferment extract, and bacillus ferment, targeting the enhancement of skin hydration, barrier function, and structural integrity. In SLS-stimulated 3D skin models and clinical settings, the cream significantly improved the expression of key barrier proteins such as filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), while reducing inflammatory markers like IL-1α, TNF-α, and PGE2. Notably, the cream facilitated a significant increase in epidermal thickness and improved the dermal–epidermal junction index (DEJI), as observed through TPM, indicating profound skin repair and enhanced barrier functionality. Clinical trials further demonstrated the cream’s reparative effects, significantly reducing symptoms in participants with sensitive skin and post-intense pulsed light (IPL) treatment scenarios. This study highlights the utility of TPM as a groundbreaking tool in cosmetic dermatology, offering real-time analysis of the effects of skincare products on skin structure and function. Full article

Ternary metal chalcogenide quantum dots (QDs), such as CuInS₂, have attracted significant attention due to their lower toxicity compared to binary counterparts containing cadmium or lead, making them promising candidates for biomedical imaging and solar energy applications. The surfactant choice is critical for controlling nanocrystal nucleation, growth kinetics, and functionalization. This directly affects the toxicity and applications of QDs. In this work, we report a synthesis protocol for PVP-capped CuInS₂ QDs in an aqueous solution. Using density functional theory (DFT) calculations, we predicted the coordination patterns of PVP on the CuInS₂ QDs surface, providing insights into the stabilization mechanism. The synthesized QDs were characterized using TEM, XRD, XPS, and FTIR to assess their morphology, chemical composition, and surface chemistry. The QDs exhibited dual photoluminescence (PL) maxima at 550 nm and 680 nm, attributed to defect-related emissions, making them suitable for cell imaging applications. Cytotoxicity studies and cell imaging experiments demonstrate the excellent biocompatibility and effective staining capabilities of the PVP-capped CuInS₂ QDs, highlighting their potential as fluorescent probes for long-term, multicolor cell imaging including two-photon microscopy. Full article

Background: Fluorocitrate (FC) ranging from 5 μM to 5 mM is often used as a specific metabolic inhibitor of the astrocytes to study astrocytic functions. Whether FC at such concentrations may affect neuronal metabolism and function in vivo remains unclear. Methods: We examined the effects of FC on the ATP levels and Ca²⁺ activity of the astrocytes and neurons in the motor cortices of living mice using two-photon microscopy. Results: We found that 25 μM and 250 μM of FC decreased the intracellular ATP levels and Ca²⁺ activity in the astrocytes in the motor cortex. Equally, 250 μM of FC, but not 25 μM of FC, reduced the intracellular ATP levels in the dendritic processes of the layer 5 pyramidal neurons. However, 25 μM of FC increased the neuronal Ca²⁺ activity, whereas ≥250 μM of FC decreased it. To test whether the differential effects of FC on neuronal Ca²⁺ activity reflect the direct effect of FC on the neurons or its indirect effect on the astrocytes, we used the CNO-hM3Dq chemogenetic approach to block astrocytic Ca²⁺ activity and examined the effect of FC. In the absence of astrocytic Ca²⁺ activity, 25 μM of FC still increased and ≥250 μM of FC reduced the dendritic Ca²⁺ activity of the neurons, respectively, suggesting a direct effect of 250 μM of FC on inhibiting neuronal Ca²⁺ activity. Further, 250 μM, but not 25 μM, of FC increased the size of the dendritic spines over 2 h. Conclusions: Our findings suggest that FC at high concentrations (≥250 μM) is not a specific inhibitor of astrocytic functions, as it directly affects neuronal metabolism and synaptic plasticity in vivo. Full article

ATTO 565, a Rhodamine-type dye, has garnered significant attention due to its remarkable optical properties, such as a high fluorescence quantum yield, and the fact that it is a relatively stable structure and has low biotoxicity. ATTO 565 has found extensive applications in combination with microscopy technology. In this review, the chemical and optical properties of ATTO 565 are introduced, along with the principles behind them. The functionality of ATTO 565 in confocal microscopy, stimulated emission depletion (STED) microscopy, single-molecule tracking (SMT) techniques, two-photon excitation–stimulated emission depletion microscopy (TPE-STED) and fluorescence correlation spectroscopy (FCS) is discussed. These studies demonstrate that ATTO 565 plays a crucial role in areas such as biological imaging and single-molecule localization, thus warranting further in-depth investigations. Finally, we present some prospects and concepts for the future applications of ATTO 565 in the fields of biocompatibility and metal ion detection. This review does not include theoretical calculations for the ATTO 565 molecule. 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

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC₁₈(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage. Full article

Two-photon excitation microscopy (TPM) and multiphoton fluorescence microscopy (MPM) are advanced forms of intravital high-resolution functional microscopy techniques that allow for the imaging of dynamic molecular processes and resolve features of the biological tissues of interest. Due to the cornea’s optical properties and the uniquely accessible position of the globe, it is possible to image cells and tissues longitudinally to investigate ocular surface physiology and disease. MPM can also be used for the in vitro investigation of biological processes and drug kinetics in ocular tissues. In corneal immunology, performed via the use of TPM, cells thought to be intraepithelial dendritic cells are found to resemble tissue-resident memory T cells, and reporter mice with labeled plasmacytoid dendritic cells are imaged to understand the protective antiviral defenses of the eye. In mice with limbal progenitor cells labeled by reporters, the kinetics and localization of corneal epithelial replenishment are evaluated to advance stem cell biology. In studies of the conjunctiva and sclera, the use of such imaging together with second harmonic generation allows for the delineation of matrix wound healing, especially following glaucoma surgery. In conclusion, these imaging models play a pivotal role in the progress of ocular surface science and translational research. Full article

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures. 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

We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection. 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