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.

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.

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.

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.

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.

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.

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.

The third-order nonlinear optical responses of zinc porphyrin oligomers: Cycles vs linear chains.

Porphyrins are widely used as potential nonlinear optical (NLO) materials because of their highly delocalized π electrons and feasible synthesis and functionalization with broad biological applications. A variety of linear and cyclic porphyrin derivatives have been synthesized, and the correlation between their structures and NLO properties awaits being disclosed. In this work, the electronic structures and third-order NLO properties of linear and cyclic butadiyne-linked zinc porphyrin oligomers have been studied by quantum chemical methods and sum-over-states model. The static second hyperpolarizability (<γ0>) increases exponentially with the number of zinc porphyrin units ([<γ0>n] = 0.67[<γ0>1]n2.63, n = 2 âˆ¼ 6) in linear π-conjugated oligomers, and the <γ0> of the linear hexamer is about 74 times that of the monomer. Such enhancement of <γ0> in linear oligomers originates from closely-lying frontier molecular orbitals available for low energy electron excitations and strong charge transfer-based excitations across porphyrins. The <γ0>s of cyclic porphyrins are lower than that of the linear hexamer, though the interaction between the ring and the ligand enhances the <γ0> of some cyclic zinc porphyrin complexes. The large two-photon absorption cross sections confer on these zinc porphyrin derivatives excellent candidates for two-photon absorption applications.

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.

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.

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.

Optical constraints on two-photon voltage imaging.

Significance: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of one-photon (1P) versus two-photon (2P) voltage imaging are not well characterized. Aim: We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach: We measure the brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in the mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results: Compared with 1P excitation, 2P excitation requires ∼ 10 4 -fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in the mouse cortex with a target SNR of 10 (spike height to baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of > 300 µ m , 2P voltage imaging using an 80-MHz source can record from no more than ∼ 12 neurons simultaneously. Conclusions: Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at a depth of > 300 µ m will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging.

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.

Two-photon Clusteroluminescence Enabled by Through-Space Conjugation for In Vivo Bioimaging.

Clusteroluminescence (CL) materials without largely conjugated structures have gained significant attention due to their unique photophysical properties and potential in bioimaging. However, low luminescence efficiency and short emission wavelength limit their development. This work designs three luminogens with CL properties (CLgens) by introducing n-electron-involved through-space conjugation (TSC) into diarylmethane. Apart from single-photon excited long-wavelength (688 nm) and high-efficiency (29%) CL, two-photon clusteroluminescence (TPCL) is successfully achieved in such small luminogens with only two isolated heteroatomic units. TSC stabilized in the aggregate state has been proven to realize efficient spatial electron delocalization similar to conventionally conjugated compounds. Encouraged by the excellent TPCL properties, two-photon imaging of blood vessels in vivo and biocompatibility verification utilizing CLgens are also achieved. This work illustrates the essential role of TSC in promoting nonlinear optical properties of CLgens and may facilitate further design and development of the next generation of bioprobes with excellent biocompatibility.

Progress in Structural and Functional In Vivo Imaging of Microglia and Their Application in Health and Disease.

The first line of defense for the central nervous system (CNS) against injury or disease is provided by microglia. Microglia were long believed to stay in a dormant/resting state, reacting only to injury or disease. This view changed dramatically with the development of modern imaging techniques that allowed the study of microglial behavior in the intact brain over time, to reveal the dynamic nature of their responses. Over the past two decades, in vivo imaging using multiphoton microscopy has revealed numerous new functions of microglia in the developing, adult, aged, injured, and diseased CNS. As the most dynamic cells in the brain, microglia continuously contact all structures and cell types, such as glial and vascular cells, neuronal cell bodies, axons, dendrites, and dendritic spines, and are believed to play a central role in sculpting neuronal networks throughout life. Following trauma, or in neurodegenerative or neuroinflammatory diseases, microglial responses range from protective to harmful, underscoring the need to better understand their diverse roles and states in different pathological conditions. In this chapter, we introduce multiphoton microscopy and discuss recent advances in structural and functional imaging technologies that have expanded our toolbox to study microglial states and behaviors in new ways and depths. We also discuss relevant mouse models available for in vivo imaging studies of microglia and review how such studies are constantly refining our understanding of the multifaceted role of microglia in the healthy and diseased CNS.

Quantification of Photoreceptors' Changes in a Diabetic Retinopathy Model with Two-Photon Imaging Microscopy.

Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR), preceding the development of microvascular abnormalities. Here, we assessed the impact of neuroinflammation on the retina of diabetic-induced rats. For this aim we have used a two-photon microscope to image the photoreceptors (PRs) at different eccentricities in unstained retinas obtained from both control (N = 4) and pathological rats (N = 4). This technique provides high-resolution images where individual PRs can be identified. Within each image, every PR was located, and its transversal area was measured and used as an objective parameter of neuroinflammation. In control samples, the size of the PRs hardly changed with retinal eccentricity. On the opposite end, diabetic retinas presented larger PR transversal sections. The ratio of PRs suffering from neuroinflammation was not uniform across the retina. Moreover, the maximum anatomical resolving power (in cycles/deg) was also calculated. This presents a double-slope pattern (from the central retina towards the periphery) in both types of specimens, although the values for diabetic retinas were significantly lower across all retinal locations. The results show that chronic retinal inflammation due to diabetes leads to an increase in PR transversal size. These changes are not uniform and depend on the retinal location. Two-photon microscopy is a useful tool to accurately characterize and quantify PR inflammatory processes and retinal alterations.

Neurofibrillary tangle-bearing neurons have reduced risk of cell death in mice with Alzheimer's pathology.

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer's disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer's donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer's.