Two-photon Microscopy Research Articles

Pseudomonas aeruginosa infection induces intragraft lymphocytotoxicity that triggers lung transplant antibody-mediated rejection

How pathogens inhibit transplant tolerance remains unclear. Here, we found that Pseudomonas aeruginosa infection, but not other common bacterial respiratory infections, increases antibody-mediated rejection (AMR) risk in recipients of lung transplants. To explore this relationship, we performed orthotopic lung transplants in mice, infected recipients with P. aeruginosa , and observed for the development of AMR. Intravital two-photon microscopy showed that P. aeruginosa rapidly invaded bronchial-associated lymphoid tissues, which resulted in acute lymphocytotoxicity, including the death of forkhead box P3 (Foxp3) + CD4 + T cells that are required to suppress AMR. P. aeruginosa –mediated AMR required expression of the type III secretion system (T3SS), which injects exotoxins into the cell cytoplasm. Through a combination of mutagenesis and epitope tagging experiments, we revealed that T3SS exotoxin T ADP ribosyl-transferase activity was sufficient for graft-resident Foxp3 + CD4 + T cell apoptosis, leading to myeloid differentiation primary response 88 (Myd88)–dependent generation of T-box expressed in T cells (T-bet)– and C-X-C motif chemokine receptor 3 (CXCR3)–positive germinal center and memory B cells with high donor antigen avidity. We also found that T-bet + and CXCR3 + B cells were elevated in biopsies from recipients of lung transplants who were diagnosed with AMR. In mice, CXCR3 deficiency restricted to B cells or CXCR3 blockade prevented AMR despite P. aeruginosa infection. Our work has identified a previously unrecognized role of bacterial virulence in lung allograft rejection and suggests potential strategies to prevent AMR for those at high risk of P. aeruginosa infection after transplant.

Evaluation of safe exposure time for two-photon microscopy imaging of acrylic-painted mock-ups

Abstract Nonlinear optical microscopies are widely used in the biological and biomedical fields, as they are non-invasive techniques that permit the safe structural and morphological characterisation of cells and tissues. They are increasingly being used in the Cultural Heritage field because of their ability to overcome some limits of the well-established optical techniques. However, since nonlinear optical microscopies use pulsed laser sources with high peak power, their application in Cultural Heritage raises concerns due to artworks’ unique, priceless, and delicate nature. In this paper, we present a new method for evaluating the photo-induced damage when using a near-infrared femtosecond pulsed laser to perform two-photon excited fluorescence imaging of acrylic-painted mock-ups. In particular, we explore herein several irradiation conditions, varying the exposure time and excitation power, in order to provide useful experimental indications for safely imaging acrylic paints with two-photon microscopy.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Accelerating biopharmaceutical cell line selection with label-free multimodal nonlinear optical microscopy and machine learning

The selection of high-performing cell lines is crucial for biopharmaceutical production but is often time-consuming and labor-intensive. We investigated label-free multimodal nonlinear optical microscopy for non-perturbative profiling of biopharmaceutical cell lines based on their intrinsic molecular contrast. Employing simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy with fluorescence lifetime imaging microscopy (FLIM), we characterized Chinese hamster ovary (CHO) cell lines at early passages (0–2). A machine learning (ML)-assisted analysis pipeline leveraged high-dimensional information to classify single cells into their respective lines. Remarkably, the monoclonal cell line classifiers achieved balanced accuracies exceeding 96.8% as early as passage 2. Correlation features and FLIM modality played pivotal roles in early classification. This integrated optical bioimaging and machine learning approach presents a promising solution to expedite cell line selection process while ensuring identification of high-performing biopharmaceutical cell lines. The techniques have potential for broader single-cell characterization applications in stem cell research, immunology, cancer biology and beyond.

A fluorescent protein C-terminal fusion knock-in is functional with TRPA1 but not TRPC5.

Transgenic mice with fluorescent protein (FP) reporters take full advantage of new in vivo imaging technologies. Therefore, we generated a TRPC5- and a TRPA1-reporter mouse based on FP C-terminal fusion, providing us with better alternatives for studying the physiology, interaction and coeffectors of these two TRP channels at the cellular and tissue level. We generated transgenic constructs of the murine TRPC5- and TRPA1-gene with a 3*GGGGS linker and C-terminal fusion to mCherry and mTagBFP, respectively. We microinjected zygotes to generate reporter mice. Reporter mice were examined for visible fluorescence in trigeminal ganglia with two-photon microscopy, immunohistochemistry and calcium imaging. Both TRPC5-mCherry and TRPA1-mTagBFP knock-in mouse models were successful at the DNA and RNA level. However, at the protein level, TRPC5 resulted in no mCherry fluorescence. In contrast, sensory neurons derived from the TRPA1-reporter mice exhibited visible mTag-BFP fluorescence, although TRPA1 had apparently lost its ion channel function. Creating transgenic mice with a TRP channel tagged at the C-terminus with a FP requires detailed investigation of the structural and functional consequences in a given cellular context and fine-tuning the design of specific constructs for a given TRP channel subtype. Different degrees of functional impairment of TRPA1 and TRPC5 constructs suggest a specific importance of the distal C-terminus for the regulation of these two channels in trigeminal neurons.

Abstract TP361: Type 2 Diabetes Enhances Leukocytes And Brain Endothelial Cells Adhesion Interactions Following Ischemic Stroke

Introduction: Stroke is a leading cause of death and disability worldwide, with diabetes being a significant risk factor that exacerbates stroke outcomes. While previous studies have identified various cell populations characteristic of stroke and other neurological diseases through transcriptomic analysis of brain and blood cells, the interaction between leukocytes and brain endothelial cells during stroke remains inadequately understood. This study aimed to investigate these intercellular interactions in the context of diabetes and stroke. Methods: We utilized a permanent ischemic stroke model in leptin receptor-deficient db/db mice (a model for type 2 diabetes) and their non-diabetic counterparts, db/+ mice. Three days post-stroke, brain cells and circulating leukocytes were isolated and subjected to single-cell RNA sequencing (scRNAseq). The CellChat tool was employed to analyze the intercellular communication between these cells. Additionally, in vivo two-photon microscopy was used to observe real-time interactions between leukocytes and brain endothelial cells in cortical veins. Results: scRNAseq analysis revealed that diabetic mice had a higher proportion of myeloid cells in the blood compared to controls. Post-stroke, both diabetic and non-diabetic mice showed an increased presence of microglia and macrophages in the brain. CellChat analysis indicated that both diabetes and stroke significantly elevated the number and strength of cell-cell interactions, with the highest levels observed in the diabetic stroke group. Notably, interactions involving the integrin family and ICAM/VCAM, known for their roles in cell adhesion between blood leukocytes and brain endothelial cells, were particularly prominent in the diabetic mice. In vivo imaging confirmed significant leukocyte accumulation in the cortical veins immediately after stroke in both genotypes, with delayed clearance observed in the diabetic group. Conclusion: The findings suggest that diabetes exacerbates the inflammatory response in stroke by enhancing leukocyte-endothelial interactions, leading to prolonged leukocyte retention in the cerebral vasculature, which likely contributes to increased inflammation and worsened stroke outcomes in diabetic conditions.

APOE4 and sedentary lifestyle synergistically impair neurovascular function in the visual cortex of awake mice

Reduced cerebral blood flow occurs early in the development of Alzheimer’s disease (AD), but the factors producing this reduction are unknown. Here, we ask whether genetic and lifestyle risk factors for AD—the ε4 allele of the Apolipoprotein (APOE) gene, and physical activity—can together produce this reduction in cerebral blood flow which leads eventually to AD. Using in vivo two-photon microscopy and haemodynamic measures, we record neurovascular function from the visual cortex of physically active or sedentary mice expressing APOE3 and APOE4 in place of murine APOE. Energy supply and demand are mismatched in APOE4 mice, with smaller increases in cerebral blood flow, blood volume and blood oxygenation occurring during neuronal activation as blood vessels frequently fail to dilate. Exercise dose-dependently overall improves neurovascular function, with an increased impact of exercise apparent after longer exposure times. Several haemodynamic measures show a larger beneficial effect of exercise in APOE4 vs. APOE3 mice. Thus, APOE4 genotype in conjunction with sedentary behaviour produces the worst neurovascular function. Promotion of physical activity may therefore be particularly important to improve cerebrovascular function and reduce dementia risk in APOE4 carriers.

Long-term live-cell imaging of GFAP+ astroglia and laminin+ vessels in organotypic mouse brain slices using microcontact printing

Organotypic brain slices are three-dimensional, 150-μm-thick sections derived from postnatal day 10 mice that can be cultured for several weeks in vitro. However, these slices pose challenges for live-cell imaging due to their thickness, particularly without access to expensive two-photon microscopy. In this study, we present an innovative method to label and visualize specific brain cell populations in living slices. Using microcontact printing, antibodies are applied directly onto the slices in a controlled 400-μm-diameter pattern. Astrocytes are labeled with glial fibrillary acidic protein (GFAP), and vessels are labeled with laminin. Subsequently, slices are incubated with secondary fluorescent antibodies (green fluorescent Alexa-488 or red fluorescent Alexa-546) and visualized using an inverted fluorescence microscope. This approach offers a cost-effective and detailed visualization technique for astroglia and vessels in living brain slices, enabling investigation to be conducted over several weeks.

Active axial motion compensation in multiphoton-excited fluorescence microscopy.

In living organisms, the natural motion caused by heartbeat, breathing, or muscle movements leads to the deformation of tissue caused by translation and stretching of the tissue structure. This effect results in the displacement or deformation of the plane of observation for intravital microscopy and causes motion-induced aberrations of the resulting image data. This, in turn, places severe limitations on the time during which specific events can be observed in intravital imaging experiments. These limitations can be overcome if the tissue motion can be compensated such that the plane of observation remains steady. We have developed a mathematical shape space model that can predict the periodic motion of a cylindrical tissue phantom resembling blood vessels. This model is then used to rapidly calculate the future position of the plane of observation of a two-photon laser scanning fluorescence microscope. The focal plane is continuously adjusted to the calculated position with a piezo-actuated objective lens holder. We demonstrate active motion compensation for non-harmonic axial displacements of the vessel phantom with a field of view up to 400 µm × 400 µm, vertical amplitudes of more than 100 µm, and at a rate of 0.5 Hz.

Disclosing long-term tolerance, efficacy and penetration properties of hyaluronic acid-coated latanoprost-loaded liposomes as chronic glaucoma therapy.

Frequent topical administration of hypotensive eye drops in glaucoma patients may lead to the development of dry eye disease (DED) symptoms, because of tear film destabilization and the adverse effects associated with antiglaucoma formulations. To address all this, in the current study preservative-free latanoprost-loaded (0.005% w/v) synthetic phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine 0.75% w/v, 1,2-dimyristoyl-sn-glycero-3-phosphocholine 0.25% w/v) liposomes dispersed in the mucoadhesive polymer hyaluronic acid (0.2% w/v), containing the osmoprotective ingredients betaine (0.40% w/v) and leucine (0.90% w/v) (LAT-HA-LIP), have been prepared and further characterised. Permeation and retention evaluations on a validated ex vivo porcine eye model revealed that the active metabolite latanoprost acid was quantified only starting from LAT-HA-LIP once passing conjunctiva, sclera and choroid compared to the marketed latanoprost (0.005% w/v) benchmark (MF). The liposomal formulation outperformed MF when applied to the corneal tissue. Additionally, distribution and interactions within corneal and scleral tissues were investigated by means of two-photon microscopy with liposomal formulations containing coumarin-6. Furthermore, acute and chronic tolerance studies on rabbits revealed no signs of discomfort or ocular damage. Schirmer's test, tear osmolarity, tear breakup time (TBUT) and fluorescence staining evaluated through the Oxford grading scale, were assessed as DED diagnostic parameters over a 25-day monitoring period; LAT-HA-LIP consistently maintained levels comparable to physiological solution (0.9% w/v NaCl) used as control, with a slight increase of TBUT values from day 15 (6.00±0.63s for control, 7.00±0.78s for LAT-HA-LIP at day 15, p=0.0066). A daily topical application of LAT-HA-LIP for 15 consecutive days, effectively lowered IOP in a sustained way (2.51-3.88mmHg mean IOP reduction over the 5-15-day period). These results highlight the clinical relevance of the proposed technological platform, able to provide IOP reduction during the simulated long-term administration and simultaneous ocular surface protection with potential for the treatment of glaucoma.

Signal subtraction using nanosecond optical pulses with two different pulse energies in two-photon photoacoustic microscopy (TP-PAM) with a ring-shaped acoustic transducer to evaluate precise thickness

Signal subtraction using nanosecond optical pulses with two different pulse energies in two-photon photoacoustic microscopy (TP-PAM) with a ring-shaped acoustic transducer to evaluate precise thickness

The Dose-Dependent Effects of Fluorocitrate on the Metabolism and Activity of Astrocytes and Neurons

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.

In vivo two-photon FLIM resolves photosynthetic properties of maize bundle sheath cells.

Maize (Zea mays L.) performs highly efficient C4 photosynthesis by dividing photosynthetic metabolism between mesophyll and bundle sheath cells. In vivo physiological measurements are indispensable for C4 photosynthesis research as photosynthetic activities are easily interrupted by leaf section or cell isolation. Yet, direct in vivo observation regarding bundle sheath cells in the delicate anatomy of the C4 leaf is still challenging. In the current work, we used two-photon fluorescence-lifetime imaging microscopy (two-photon-FLIM) to access the photosynthetic properties of bundle sheath cells on intact maize leaves. The results provide spectroscopic evidence for the diminished total PSII activity in bundle sheath cells at its physiological level and show that the single PSIIs could undergo charge separation as usual. We also report an acetic acid-induced chlorophyll fluorescence quenching on intact maize leaves, which might be a physiological state related to the nonphotochemical quenching mechanism.

Carbazole-Based Eu3+ Complexes for Two-Photon Microscopy Imaging of Live Cells.

Lanthanide(III) complexes with two-photon absorbing antennas are attractive for microscopy imaging of live cells because they can be excited in the NIR. We describe the synthesis and luminescence and imaging properties of two Eu3+ complexes, mTAT[Eu·L-CC-Ar-Cz] and mTAT[Eu·L-Ar-Cz], with (N-carbazolyl)-aryl-alkynyl-picolinamide and (N-carbazolyl)-aryl-picolinamide antennas, respectively, conjugated to the TAT cell-penetrating peptides. Contrary to what was previously observed with related Eu3+ complexes with carbazole-based antennas in a mixture of water and organic solvents, these two complexes show very low emission quantum yield (ΦEu < 0.002) in purely aqueous buffers. A detailed spectroscopic study on mTAT[Eu·L-Ar-Cz] reveals that the quantum yield of emission is strongly polarity dependent─the less polar the medium, the higher the quantum yield─and that the emission quenching in water is likely due to a photoinduced electron transfer between the excited carbazole-based antenna and Eu3+ that efficiently competes with the energy transfer process. Nevertheless, mTAT[Eu·L-Ar-Cz] shows a significant two-photon cross-section of 100 GM at 750 nm, which is interesting for two-photon microscopy. The live cell imaging properties of mTAT[Eu·L-Ar-Cz] and the two other conjugates were investigated. Cytosolic delivery was clearly evidenced in the case of mTAT[Eu·L-Ar-Cz] when cells are coincubated with this compound and a nonluminescent dimeric TAT derivative, dFFLIPTAT.

Long-term excessive salt consumption alters villous and crypt morphology and the protein expression of uroguanylin, TRPV6 and PMCA1b in the rat small intestine.

Although long-term high dietary sodium consumption often aggravates hypertension and bone loss, sodium in the intestinal lumen has been known to promote absorption of nutrients and other ions, e.g., glucose and calcium. However, whether high-salt diet (HSD) altered mucosal morphology, villous cell turnover and calcium transporter expression remained elusive. Herein, rats were treated with HSD containing 8% wt/wt NaCl for up to 5 months. HSD rats exhibited a marked increase in sodium intake with high fecal and urinary sodium excretion, as compared to the control group treated with normal diet. Intestinal histomorphometry revealed increasing of crypt depth and villous height in 3- and 4-month HSD groups, respectively, consistent with larger mucosal-to-serosal amplification ratio that reflected an increased surface area for nutrient absorption. The signals of Ki-67-positive cells was enhanced in the crypts as visualized by multiphoton fluorescence microscopy, whereas the TUNEL-positive cells were decreased in the villi of HSD, suggesting greater crypt cell proliferation and a reduction of villous cell apoptosis. Confocal microscopy showed higher expression of TRPV6 protein in the villous tip of HSD, while PMCA1 expression was increased in villous tip and crypt areas. The percentage of cells with highly expressed uroguanylin-an endogenous intestinal natriuretic peptide-was significantly higher in HSD group. In conclusion, HSD profoundly changed the intestinal morphology and turnover of epithelial cell, increased the expression of calcium transporters and uroguanylin. Our findings reflect pathophysiological adaptations in the intestine, which might be another target organ for drug discovery against HSD-induced osteopathy in the future.

Incomplete remyelination via therapeutically enhanced oligodendrogenesis is sufficient to recover visual cortical function

Myelin loss induces neural dysfunction and contributes to the pathophysiology of neurodegenerative diseases, injury conditions, and aging. Because remyelination is often incomplete, better understanding endogenous remyelination and developing remyelination therapies that restore neural function are clinical imperatives. Here, we use in vivo two-photon microscopy and electrophysiology to study the dynamics of endogenous and therapeutic-induced cortical remyelination and functional recovery after cuprizone-mediated demyelination in mice. We focus on the visual pathway, which is uniquely positioned to provide insights into structure-function relationships during de/remyelination. We show endogenous remyelination is driven by recent oligodendrocyte loss and is highly efficacious following mild demyelination, but fails to restore the oligodendrocyte population when high rates of oligodendrocyte loss occur quickly. Testing a thyromimetic (LL-341070) compared to clemastine, we find it better enhances oligodendrocyte gain and hastens recovery of neuronal function. The therapeutic benefit of the thyromimetic is temporally restricted, and it acts exclusively following moderate to severe demyelination, eliminating the endogenous remyelination deficit. However, we find regeneration of oligodendrocytes and myelin to healthy levels is not necessary for recovery of visual neuronal function. These findings advance our understanding of remyelination and its impact on functional recovery to inform future therapeutic strategies.

Establishing an animal model for post-inflammatory hyperpigmentation following fractional CO2 laser application.

Post-inflammatory hyperpigmentation (PIH) is a common cosmetic concern, often leading to significant psychological distress for the patients. With the widespread application of lasers including ablative fractional resurfacing (AFR) with a 10,600nm CO2 laser, PIH caused by lasers is becoming increasingly common. But due to the absence of an appropriate animal research model, our understanding of pathophysiological mechanisms and preventive strategies for PIH remains limited. This study aimed to establish an animal model to investigate PIH following AFR CO2 laser application, focusing on the dynamic changes in melanin, inflammatory cytokines, growth factors, and skin structures as PIH developed. We employed pigmented guinea pigs as our experimental subjects and conducted our research in two phases. In the first phase, we utilized three modes of AFR CO2 laser to identify which laser mode could induce PIH by monitoring dynamic melanin changes. In the second phase, the laser mode that most reliably induced PIH was applied to re-establish the PIH model. Pathophysiological changes during PIH progression were investigated through histopathological observations, real-time quantitative polymerase chain reaction, and two-photon microscopy. We successfully established a replicable animal model for PIH following AFR CO2 laser application. We observed a significant increase in inflammatory cytokines and growth factors within the skin tissue by the second week, with stable pigmentation becoming apparent by the third week. Our research provides a promising animal model for understanding and further investigating the mechanisms of PIH after laser procedures. V (animal study).

Evaluation of safe exposure time for two-photon microscopy imaging of acrylic-painted mock-ups

Abstract Nonlinear optical microscopies are widely used in the biological and biomedical fields, as they are non-invasive techniques that permit the safe structural and morphological characterisation of cells and tissues. They are increasingly being used in the Cultural Heritage field because of their ability to overcome some limits of the well-established optical techniques. However, since nonlinear optical microscopies use pulsed laser sources with high peak power, their application in Cultural Heritage raises concerns due to artworks' unique, priceless, and delicate nature. In this paper, we present a new method for evaluating the photo-induced damage when using a near-infrared femtosecond pulsed laser to perform two-photon excited fluorescence imaging of acrylic-painted mock-ups. In particular, we explore herein several irradiation conditions, varying the exposure time and excitation power, in order to provide useful experimental indications for safely imaging acrylic paints with two-photon microscopy.

Compact module for video-rate image mosaics in two-photon microscopy.

Biological applications using multiphoton microscopy increasingly seek a larger field of view while maintaining sufficient temporal sampling to observe dynamic biological processes. Multiphoton imaging also requires high numerical aperture microscope objectives to realize efficient non-linear excitation and collection of fluorescence. This combination of low-magnification and high-numerical aperture poses a challenge for system design. To address this, the use of a liquid crystal polarization grating stack is proposed here to temporally sequence through multiple fields of view. This solution pans the native field of view with minimal latency and zero inertial movement of either the microscope or biological sample. Implemented as a simple add-on unit to existing multi-photon microscopes, this device increases the total field size by 4x, covering up to 7.6mm2. Performance constraints and functional demonstration of imaging neural activity are presented.