Organic two-photon-absorbing photosensitizers can overcome competing light absorption in organic photocatalysis.

Conventional organic photocatalysis typically relies on ultraviolet and short-wavelength visible photons as the energy source. However, this approach often suffers from competing light absorption by reactants, products, intermediates, and co-catalysts, leading to reduced quantum efficiency and side reactions. To address this issue, we developed novel organic two-photon-absorbing (TPA) photosensitizers capable of functioning under deep red and near-infrared light irradiation. Three model reactions including cyclization, Sonogashira Csp2-Csp cross-coupling, and Csp2-N cross-coupling reactions were selected to compare the performance of the new photosensitizers under both blue (427 nm) and deep red (660 nm) light irradiation. The obtained results unambiguously prove that for reactions involving blue light-absorbing reactants, products, and/or co-catalysts, deep red light source resulted in better performance than blue light when utilizing our TPA photosensitizers. This work highlights the potential of our metal-free TPA photosensitizers as a sustainable and effective solution to mitigate the competing light absorption issue in photocatalysis, not only expanding the scope of organic photocatalysts but also reducing reliance on expensive Ru/Ir/Os-based photosensitizers.

Trifluoromethyl-pyrrolidone phthalocyanine nanoparticles for targeted lipid droplet imaging and in vitro photodynamic therapy in breast cancer cells.

Lipid droplets (LDs) serve as vital subcellular organelles, crucial for the maintenance of lipid and energy homeostasis within cells. Their visualization is of significant value for elucidating the intricate interactions between LDs and other cellular organelles. Despite the importance of LDs, the literature on the utilization of phthalocyanine-based photosensitizers for targeted LD imaging and two-photon imaging-guided photodynamic therapy (PDT) remains sparse. In this study, we have designed and synthesized trifluoromethyl-pyrrolidone silicon phthalocyanine (PyCF3SiPc). To enhance the water solubility of PyCF3SiPc and improve its tumor cells accumulation, we employed 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-mPEG2000) as a nanocarrier, thereby formulating DSPE@PyCF3SiPc nanoparticles. Our in vitro experiments in MCF-7 cells demonstrated that DSPE@PyCF3SiPc selectively targets and visualizes LDs, offering a reliable tool for tracking their dynamic movement. Moreover, DSPE@PyCF3SiPc demonstrates considerable phototoxicity against MCF-7 cells subjected to PDT underscoring its potential as an effective therapeutic agent. In conclusion, DSPE@PyCF3SiPc presents itself as a promising novel probe for the dual purpose of monitoring the dynamic movement of LDs and guiding imaging-assisted PDT. The development of this nanoparticle system not only advances our understanding of LD biology but also paves the way for innovative therapeutic strategies in oncology.

Covalent assembly-based two-photon fluorescent probes for in situ visualizing nitroreductase activities: From cancer cells to human cancer tissues.

Nitroreductase (NTR) is widely regarded as a biomarker whose enzymatic activity correlates with the degree of hypoxia in solid malignant tumors. Herein, we utilized 2-dimethylamino-7-hydroxynaphthalene as fluorophore linked diverse nitroaromatic groups to obtain four NTR-activatable two-photon fluorescent probes based on covalent assembly strategy. With the help of computer docking simulation and in vitro assay, the sulfonate-based probe XN3 was proved to be able to identify NTR activity with best performances in rapid response, outstanding specificity, and sensitivity in comparison with the other three probes. Furthermore, XN3 could detect the degree of hypoxia by monitoring NTR activity in kinds of cancer cells with remarkable signal-to-noise ratios. In cancer tissue sections of the breast and liver in mice, XN3 had the ability to differentiate between healthy and tumorous tissues, and possessed excellent fluorescence stability, high tissue penetration and low tissue autofluorescence. Finally, XN3 was successfully utilized for in situ visualizing NTR activities in human transverse colon and rectal cancer tissues, respectively. The findings suggested that XN3 could directly identify the boundary between cancer and normal tissues by monitoring NTR activities, which provides a new method for imaging diagnosis and intraoperative navigation of tumor tissue.

In vivo spectrally unmixed multi-photon imaging of longitudinal axon-glia changes in injured spinal white matter.

Understanding the sequence of cellular responses and their contributions to pathomorphogical changes in spinal white matter injuries is a prerequisite for developing efficient therapeutic strategies for spinal cord injury (SCI) as well as neurodegenerative and inflammatory diseases of the spinal cord such as amyotrophic lateral sclerosis and multiple sclerosis. We have developed several types of surgical procedures suitable for acute one-time and chronic recurrent in vivo multiphoton microscopy of spinal white matter [1]. Sophisticated surgical procedures were combined with transgenic mouse technology to image spinal tissue labeled with up to four fluorescent proteins (FPs) in axons, astrocytes, microglia, and blood vessels. To clearly separate the simultaneously excited FPs, spectral unmixing including iterative procedures was performed after imaging the diversely labeled spinal white matter with a custom-made 4-channel two-photon laser-scanning microscope. In our longitudinal multicellular studies of injured spinal white matter, we imaged axonal dynamics and invasion of microglia and astrocytes for a time course of over 200 days after SCI. Our methods offer ideal platforms for investigating acute and chronic cellular dynamics, cell-cell interactions, and metabolite fluctuations in health and disease as well as pharmacological manipulations in vivo.

Capillary regression leads to sustained local hypoperfusion by inducing constriction of upstream transitional vessels.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a feature of many neurological disorders and injuries and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ~59% of the injuries resulted in regression of the capillary segment 7 to 14 d following injury, and the remaining repaired to reestablish blood flow within 7 d. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days postinjury in both awake and anesthetized mice. The degree of vasomotor dynamics was chronically attenuated in the ACT zone consequently reducing blood flow in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how focal capillary injury and regression can impair the microvascular sensory web and contribute to cerebral hypoperfusion.

Comparison of compartmental analytical Blood-Oxygen-Level-Dependent functional Magnetic Resonance Imaging models against Monte Carlo simulations performed over cortical micro-angiograms.

Blood oxygen level-dependent functional magnetic resonance imaging (BOLD fMRI) arises from a physiological and physical cascade of events taking place at the level of the cortical microvasculature which constitutes a medium with complex geometry. Several analytical models of the BOLD contrast have been developed, but these have not been compared directly against detailed bottom-up modeling methods. Using a 3D modeling method based on experimentally measured images of mice microvasculature and Monte Carlo simulations, we quantified the accuracy of two analytical models to predict the amplitude of the BOLD response from 1.5 to 7 T, for different echo time (TE) and for both gradient echo and spin echo acquisition protocols. We also showed that accounting for the tridimensional structure of the microvasculature results in more accurate prediction of the BOLD amplitude, even if the values for SO2 were averaged across individual vascular compartments. A secondary finding is that modeling the venous compartment as two individual compartments results in more accurate prediction of the BOLD amplitude compared with standard homogenous venous modeling, arising from the bimodal distribution of venous SO2 across the microvasculature in our data.

Bone-Mimetic Osteon Microtopographies on Poly-ε-Caprolactone Enhance the Osteogenic Potential of Human Mesenchymal Stem Cells.

The attributes of implant surfaces are pivotal for successful osseointegration. Among surface engineering strategies, microtopography stands out as a promising approach to promote early cellular interactions. This study aims to design and craft a novel biomimetic osteon-like surface modification and to compare its impact on human mesenchymal stem cells (hMSCs) with four established topographies: blank, inverted pyramids, protrusions, and grooves. Poly-ε-caprolactone samples are fabricated using 2-photon-polymerization and soft lithography, prior to analysis via scanning electron microscopy (SEM), water contact angle (WCA), and protein adsorption assays. Additionally, cellular responses including cell attachment, proliferation, morphology, cytoskeletal organization, and osteogenic differentiation potential are evaluated. SEM confirms the successful fabrication of microtopographies, with minimal effect on WCA and protein adsorption. Cell attachment experiments demonstrate a significant increase on the osteon-like structure, being three times higher than on the blank. Proliferation assays indicate a fourfold increase with osteon-like microtopography compared to the blank, while ALP activity is notably elevated with osteon-like microtopography at days 7 (threefold increase over blank) and 14 (fivefold increase over blank). In conclusion, the novel biomimetic osteon-like structure demonstrates favorable responses from hMSCs, suggesting potential for promoting successful implant integration in vivo.

Shot-Noise Limited Nonlinear Optical Imaging Excited With GHz Femtosecond Pulses and Denoised by Deep-Learning.

Multiphoton fluorescence microscopy excited with femtosecond pulses at high repetition rates, particularly in the range of 100's MHz to GHz, offers an alternative solution to suppress photoinduced damage to biological samples, for example, photobleaching. Here, we demonstrate the use of a U-Net-based deep-learning algorithm for suppressing the inherent shot noise of the two-photon fluorescence images excited with GHz femtosecond pulses. With the trained denoising neural network, the image quality of the representative two-photon fluorescence images of the biological samples is shown to be significantly improved. Moreover, for input raw images with even SNR reduced to -4.76 dB, the trained denoising network can recover the main image structure from noise floor with acceptable fidelity and spatial resolution. It is anticipated that the combination of GHz femtosecond pulses and deep-learning denoising algorithm can be a promising solution for eliminating the trade-off between photoinduced damage and image quality in nonlinear optical imaging platforms.

High-Resolution Structuring of Silica-Based Nanocomposites for the Fabrication of Transparent Multicomponent Glasses with Adjustable Properties.

Silicate-based multicomponent glasses are of high interest for technical applications due to their tailored properties, such as an adaptable refractive index or coefficient of thermal expansion. However, the production of complex structured parts is associated with high effort, since glass components are usually shaped from high-temperature melts with subsequent mechanical or chemical postprocessing. Here for the first time the fabrication of binary and ternary multicomponent glasses using doped nanocomposites based on silica nanoparticles and photocurable metal oxide precursors as part of the binder matrix is presented. The doped nanocomposites are structured in high resolution using UV-casting and additive manufacturing techniques, such as stereolithography and two-photon lithography. Subsequently, the composites are thermally converted into transparent glass. By incorporating titanium oxide, germanium oxide, or zirconium dioxide into the silicate glass network, multicomponent glasses are fabricated with an adjustable refractive index nD between 1.4584-1.4832 and an Abbe number V of 53.85-61.13. It is further demonstrated that by incorporating 7 wt% titanium oxide, glasses with ultralow thermal expansion can be fabricated with so far unseen complexity. These novel materials enable for the first time high-precision lithographic structuring of multicomponent silica glasses with applications from optics and photonics, semiconductors as well as sensors.

General Post-Regulation Strategy of AIEgens' Photophysical Properties for Intravital Two-Photon Fluorescence Imaging.

Fluorogens with aggregation-induced emission (AIEgens) are promising agents for two-photon fluorescence (TPF) imaging. However, AIEgens' photophysical properties are fixed and unoptimizable once synthesized. Therefore, it is urgent and meaningful to explore an efficient post-regulation strategy to optimize AIEgens' photophysical properties. Herein, a general and efficient post-regulation strategy is reported. By simply tuning the ratio of inert AIEgens within binary nanoparticles (BNPs), the fluorescence quantum yield and two-photon absorption cross-section of functional AIEgens are enhanced by 8.7 and 5.4 times respectively, which are not achievable by conventional strategies, and the notorious phototoxicity is almost eliminated. The experimental results, theoretical simulation, and mechanism analysis demonstrated its feasibility and generality. The BNPs enabled deep cerebrovascular network imaging with ≈1.10 mm depth and metastatic cancer cell detection with single-cell resolution. Furthermore, the TPF imaging quality is improved by the self-supervised denoising algorithm. The proposed binary molecular post-regulation strategy opened a new avenue to efficiently boost the AIEgens' photophysical properties and consequently TPF imaging quality.

Microvascular plasticity in mouse stroke model recovery: Anatomy statistics, dynamics measured by longitudinal in vivo two-photon angiography, network vectorization.

This manuscript quantitatively investigates remodeling dynamics of the cortical microvascular network (thousands of connected capillaries) following photothrombotic ischemia (cubic millimeter volume, imaged weekly) using a novel in vivo two-photon angiography and high throughput vascular vectorization method. The results suggest distinct temporal patterns of cerebrovascular plasticity, with acute remodeling peaking at one week post-stroke. The network architecture then gradually stabilizes, returning to a new steady state after four weeks. These findings align with previous literature on neuronal plasticity, highlighting the correlation between neuronal and neurovascular remodeling. Quantitative analysis of neurovascular networks using length- and strand-based statistical measures reveals intricate changes in network anatomy and topology. The distance and strand-length statistics show significant alterations, with a peak of plasticity observed at one week post-stroke, followed by a gradual return to baseline. The orientation statistic plasticity peaks at two weeks, gradually approaching the (conserved across subjects) stroke signature. The underlying mechanism of the vascular response (angiogenesis vs. tissue deformation), however, is yet unexplored. Overall, the combination of chronic two-photon angiography, vascular vectorization, reconstruction/visualization, and statistical analysis enables both qualitative and quantitative assessments of neurovascular remodeling dynamics, demonstrating a method for investigating cortical microvascular network disorders and the therapeutic modes of action thereof.

Two-photon excitation two-dimensional fluorescence spectroscopy (2PE-2DFS) of the fluorescent nucleobase 6-MI.

Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.

A Nano-Aggregatable Acedan Derivative for Clathrin-Mediated Cellular Uptake and Two-Photon Imaging of Diabetes-Associated Lipid Droplets.

Lipid droplets (LDs), the essential cytosolic fat storage organelles, have emerged as pivotal regulators of cellular metabolism and are implicated in various diseases. The noninvasive monitoring of LDs necessitates fluorescent probes with precise organelle selectivity and biocompatibility. Addressing this need, we have engineered a probe by strategically modifying the structure of a conventional two-photon-absorbing dipolar dye, acedan. This innovative approach induces nanoaggregate formation in aqueous environments, leading to aggregation-induced fluorescence quenching. Upon cellular uptake via clathrin-mediated endocytosis, the probe selectively illuminates within LDs through a disassembly process, effectively distinguishing LDs from the cytosol with exceptional specificity. This breakthrough enables the high-fidelity imaging of LDs in both cellular and tissue environments. In a pioneering investigation, we probed LDs in a diabetes model induced by streptozotocin, unveiling significantly heightened LD accumulation in cardiac tissues compared to other organs, as evidenced by TP imaging. Furthermore, our exploration of a lipopolysaccharide-mediated cardiomyopathy model revealed an LD accumulation during heart injury. Thus, our developed probe holds immense potential for elucidating LD-associated diseases and advancing related research endeavors.

Visual experience reduces the spatial redundancy between cortical feedback inputs and primary visual cortex neurons.

The role of experience in the organization of cortical feedback (FB) remains unknown. We measured the effects of manipulating visual experience on the retinotopic specificity of supragranular and infragranular projections from the lateromedial (LM) visual area to layer (L)1 of the mouse primary visual cortex (V1). LM inputs were, on average, retinotopically matched with V1 neurons in normally and dark-reared mice, but visual exposure reduced the fraction of spatially overlapping inputs to V1. FB inputs from L5 conveyed more surround information to V1 than those from L2/3. The organization of LM inputs from L5 depended on their orientation preference and was disrupted by dark rearing. These observations were recapitulated by a model where visual experience minimizes receptive field overlap between LM inputs and V1 neurons. Our results provide a mechanism for the dependency of surround modulations on visual experience and suggest how expected interarea coactivation patterns are learned in cortical circuits.

Effect of Diabetes on Microvascular Morphology and Permeability of Rat Skeletal Muscle: In Vivo Imaging Using Two-Photon Laser Scanning Microscopy.

This investigation evaluated the microvascular permeability and ultrastructure of skeletal muscle capillaries in skeletal muscle of diabetic (DIA) rats using two-photon laser scanning microscopy (TPLSM) and transmission electron microscopy (TEM). Microvascular permeability was assessed in the tibialis anterior muscle of control (CON) and DIA (streptozocin) male Wistar rats (n = 20, 10-14 wk) by in vivo imaging using TPLSM after fluorescent dye intravenous infusion. Fluorescent dye leakage was quantified to determine microvascular permeability. The ultrastructure was imaged by TEM ex vivo to calculate the size and number of intercellular clefts between capillary endothelial cells and also intracellular vesicles. Compared with control, the volumetrically determined interstitial fluorescent dye leakage, the endothelial cell thickness, and the number of intercellular clefts per capillary perimeter were significantly higher, and the cleft width was significantly narrower in TA of DIA (interstitial fluorescent dye leakage, 2.88 ± 1.40 vs. 10.95 ± 1.41 µm3 x min x 106; endothelial thickness 0.28 ± 0.02 vs. 0.45 ± 0.03 µm; number of intercellular clefts per capillary perimeter 6.3 ± 0.80 vs. 13.6 ± 1.7 /100 µm; cleft width 11.92 ± 0.95 vs. 8.40 ± 1.03 nm, CON vs. DIA respectively, all p <0.05). The size of intracellular vesicles in the vascular endothelium showed an increased proportion of large vesicles in the DIA group compared to the CON group (p < 0.05). Diabetes mellitus enhances the microvascular permeability of skeletal muscle microvessels, due, in part, to a higher density and narrowing of the endothelial intercellular clefts, and larger intracellular vesicles.

Surface porosity of enamel white spot lesions and penetration of fluorapatite nanocrystals into their subsurface: proof-of-concept study.

OBJECTIVES: In this study, we used atomic force microscopy (AFM) to quantify the size of surface pore apertures of enamel white spot lesions and then demonstrated the penetration of fluorapatite nanocrystals (nFA) into the subsurface of these lesions. METHODS: For the porosity study, enamel lesions were created on three sound human teeth using a demineralizing gel for 8 days. The interface between sound enamel and the artificial lesion was analyzed by AFM. To visualize the penetration of nFA tagged with a calcium-binding fluorophore (Fluo-4) into the subsurface of white spot lesions, we used two-photon microscopy. Sixteen extracted human teeth with either active, natural, or in vitro-created carious lesions in enamel were randomly divided into three groups. The teeth were treated for 2 min with either a suspension of tagged nFA crystals, Fluo-4 alone, or deionized water, and left for 30 min before being washed with distilled water and examined microscopically. RESULTS: A greater concentration of surface pores with larger areas was observed on the in vitro demineralized enamel (29 % of pores greater than 1.0 µm2) when compared with the adjacent sound enamel (8 % of pores greater than 1.0 µm2) (p=0.012, Fisher exact test). In vitro and natural lesions treated with tagged nFA showed fluorescence at depths ranging from 50 to 170 µm, demonstrating penetration of the nFA into the lesion subsurface. The lesions treated with Fluo-4 alone with no crystals showed mostly surface fluorescence (restricted to the outer 25 µm), while those treated with deionized water showed minimal (restricted to the outer 20 µm) to no fluorescence. CONCLUSION: We have demonstrated the use of AFM to quantify the surface pore apertures and two-photon microscopy to visualize nFA crystals in the subsurface of non-cavitated enamel lesions. CLINICAL SIGNIFICANCE: The restoration of the subsurface of non-cavitated caries lesions is a clinical challenge. This study demonstrated that a 2 min application of nFA could penetrate through the surface apertures of non-cavitated enamel lesions into their subsurface.

Azaacene Diradicals Based on Non-Kekulé Meta-Quinodimethane with Large Two-Photon Cross-Sections in the Infrared Spectral Region.

Non-Kekulé quinoidal azaacences m-A (1a,b) were synthesized and compared to their para- and ortho-quinodimethane analogues. m-Adisplay high diradical characters (1b: y0 = 0.88) due to their meta-quinodimethane (m-QDM) topology. Electron paramagnetic, nuclear magnetic resonance spectroscopies and supraquantum interference device measurements in combination with quantum-chemical calculations revealed singlet ground states for m-A with singlet-triplet gaps ΔEST (0.13-0.25 kcal mol-1) and thermally populated triplet states. These non-Kekulé structures are over all void of zwitterionic character and possess record high two-photon absorption cross sections over a broad spectral range in the near-infrared.

Ninjin'yoeito Modulates Baseline and Reperfusion-Induced Changes in the Arteriole Diameter and Blood Flow in the Cerebral Cortex of Anesthetized Mice.

OBJECTIVE: Intragastric administration of ninjin'yoeito (NYT), a traditional Japanese herbal medicine, reportedly prevents the decrease in baseline cerebral blood flow (CBF) in the cortex following gastric administration of water. We investigated the effect of NYT on baseline and dynamic changes in cerebral cortical arteriole diameter. METHODS: Urethane-anesthetized mice were intragastrically administered 1 g/kg NYT or distilled water (DW). The artery in the left parietal cortex was imaged using two-photon microscopy. The baseline diameter of penetrating arterioles was measured before and 50-60 min after administration. Dynamic CBF and arteriole diameter changes before, during, and after transient occlusion of the left common carotid artery were measured approximately 10 min after administration. RESULTS: DW decreased the baseline diameter of the penetrating arterioles, whereas NYT did not. During occlusion, the increase in penetrating arteriole diameter was comparable for DW and NYT; however, during reperfusion, the return to preocclusion diameter was slower for NYT than DW. Laser-speckle contrast imaging confirmed that CBF, although comparable during occlusion, was higher during reperfusion for NYT than DW. CONCLUSIONS: These results suggest that NYT attenuates vasoconstriction in penetrating arterioles after intragastric administration and during cerebral reperfusion, contributing to CBF regulation.

Emerging technologies toward the integration of multiple functionalities on non-planar implantable neurophotonics probes.

The continuous exchange between the neuroscience and neuroengineering communities that took place over the past decades has uncovered a multitude of technological solutions to interface with the brain. In this framework, a fascinating approach relies on the integration of multiple activation and monitoring capabilities in the same implantable neural probe to better study the multifaceted nature of neural signaling and related functions in the deep brain regions. We highlight current challenges and perspectives on technological developments that could potentially enable the integration of multiple functionalities on optical fiber-based non-planar implantable neurophotonics probes.