Multiphoton Microscopy Research Articles

Photo-aging evaluation - In vitro biological endpoints combined with collagen density assessment with multi-photon microscopy

BackgroundUltraviolet exposure has profound effect on the dermal connective tissue of human skin. ObjectiveWe aimed to develop and validate an evaluation method/methodology using a full-thickness reconstructed skin model, to assess the anti-photoaging efficacy of cosmetic ingredients and sunscreen formulas by blending multi relevant biological endpoints including the newly developed dermal collagen quantification method with Multi-photon microscopy. MethodsThe response of ex vivo human skin to UVA exposure was first characterized with multiphoton microscopy. Reconstructed full-thickness skin models was then used to reproduce the data and to create a proof-of-concept study by treating the models with sunscreen prototypes A or B, which differ on their UVA absorption properties, and systemic Vitamin C (Vit C). After exposure to UVA, the collagen density was quantified via multiphoton microscopy with automatic imaging processing. Histology, fibroblasts number, metalloprotease 1 (MMP1) secretion were also assessed. ResultsUVA exposure induced pronounced reduction in collagen density and increased MMP1 secretion within both ex vivo human skin and reconstructed skin. Histological damage and fibroblast disappearance was observed with reconstructed skin. Within the proof-of-concept study prototype B, possessing higher UVA filtration, gave better protection than prototype A on the UV associated biological markers, and association with Vit C boosted sunscreen formula efficacy. ConclusionsThe photoaging evaluation method, consists of multi biological markers as well as dermal collagen quantification, is a relevant mean to assess the pre-clinical efficacy of anti-photoaging ingredients and sunscreen products. This approach is also beneficial for evaluating the efficacy of sunscreens and photoprotective ingredients.

Disruptions in sleep‐dependent brain rhythms contribute to Alzheimer’s pathogenesis

Sleep-dependent brain rhythms are important for memory consolidation during sleep, and Alzheimer's disease (AD) patients experience memory disturbances. We sought to examine sleep-dependent brain rhythms, slow oscillations, in an animal model of AD (APP mice). The power of slow oscillations was decreased early in the disease progression. Soluble amyloid-beta was sufficient to disrupt the slow waves. Cortical GABA levels were low in APP mice and application of exogenous GABA restored the slow oscillations, indicating that aberrant excitatory activity within the cortical circuit was responsible for slow oscillation dysfunction. Next we sought to manipulate slow waves in APP mice with optogenetics. Driving slow oscillations at normal frequency with light activation of channelrhodopsin-2 (ChR2) expressed in cortical neurons restored slow wave power by synchronizing neuronal activity. Using multiphoton microscopy, we performed longitudinal imaging of senile plaques and monitored intracellular calcium. Cytosolic calcium is a surrogate marker of neuronal activity and is normally tightly regulated. We had previously demonstrated that resting calcium levels measured with the genetically encoded calcium sensor YellowCameleon 3.6 were elevated in a subset of neurons in APP transgenics, and hypothesized that an effective treatment would restore calcium to control levels. Driving slow oscillation activity with optogenetics halted amyloid plaque deposition and prevented calcium overload associated with this pathology. On the other hand, driving slow oscillation activity at twice the normal frequency resulted in increased amyloid production, increased amyloid plaque deposition, disruptions in neuronal calcium homeostasis, and loss of synaptic spines. Therefore, while restoration of physiological circuit dynamics is sufficient to abrogate the progression of Alzheimer's disease pathology and should be considered an avenue for clinical treatment of patients with sleep disorders, pathophysiological stimulation of neuronal circuits leads to activity dependent acceleration of amyloid production, aggregation and downstream neuronal dysfunction.

Visualizing polymeric components that define distinct root barriers across plant lineages.

ABSTRACTHydrophobic cell wall depositions in roots play a key role in plant development and interaction with the soil environment, as they generate barriers that regulate bidirectional nutrient flux. Techniques to label the respective polymers are emerging, but are efficient only in thin roots or sections. Moreover, simultaneous imaging of the barrier constituents lignin and suberin remains problematic owing to their similar chemical compositions. Here, we describe a staining method compatible with single- and multiphoton confocal microscopy that allows for concurrent visualization of primary cell walls and distinct secondary depositions in one workflow. This protocol permits efficient separation of suberin- and lignin-specific signals with high resolution, enabling precise dissection of barrier constituents. Our approach is compatible with imaging of fluorescent proteins, and can thus complement genetic markers or aid the dissection of barriers in biotic root interactions. We further demonstrate applicability in deep root tissues of plant models and crops across phylogenetic lineages. Our optimized toolset will significantly advance our understanding of root barrier dynamics and function, and of their role in plant interactions with the rhizospheric environment.

Imaging the different timescales of germinal center selection.

Germinal centers (GCs) are the site of antibody affinity maturation, a fundamental immunological process that increases the potency of antibodies and thereby their ability to protect against infection. GC biology is highly dynamic in both time and space, making it ideally suited for intravital imaging. Using multiphoton laser scanning microscopy (MPLSM), the field has gained insight into the molecular, cellular, and structural changes and movements that coordinate affinity maturation in real time in their native environment. On the other hand, several limitations of MPLSM have had to be overcome to allow full appreciation of GC events taking place across different timescales. Here, we review the technical advances afforded by intravital imaging and their contributions to our understanding of GC biology.

Acid-induced gels from soy and whey protein thermally-induced mixed aggregates: Rheology and microstructure

In this study, we explored how substituting whey protein isolate (WPI) with soy protein isolate (SPI) affects the linear and non-linear rheological behavior of acid-induced gels, and their microstructures. Commercial SPI and WPI dispersions (pH 7.0, 3.0 mS/cm) were preheated (95 °C, 30 min) at different protein concentrations (2%, 4%, 6%, and 8% w/w) and SPI: WPI ratios (0: 4, 1: 3, 2: 2, 3: 1 and 4: 0). The resultant thermally-induced aggregates were characterized before gelation was induced by glucono-δ-lactone (GDL). Small and large amplitude oscillatory shear (SAOS and LAOS) tests showed that replacing WPI with SPI decreased the strength (lower G′) and stretchability (lower γc) of acid-induced gels in the linear viscoelastic (LVE) regime. Gels containing SPI behaved more similar to pure SPI gels in the non-linear viscoelastic (NLVE) regime: displaying a relatively elastic response at large strain and a gradual transition to plastic behavior. The changes in rheological properties were explained by the differences in the gel microstructures, via fractal scaling theory, multiphoton laser scanning microscopy (MLSM) and scanning electron microscopy (SEM). WPI gels formed denser and homogenous gel networks with very strong inter-floc links, while hybrid gels and pure SPI gels formed coarser and more porous networks with intermediate inter-floc links. The constituent flocs in the latter were larger, with rougher, more elongated and branched structures. The present results provide useful information for future attempts to replace WPI with SPI in food products based on acid-induced gelation.

Human Epidermal Zinc Concentrations after Topical Application of ZnO Nanoparticles in Sunscreens.

Zinc oxide nanoparticle (ZnO NP)-based sunscreens are generally considered safe because the ZnO NPs do not penetrate through the outermost layer of the skin, the stratum corneum (SC). However, cytotoxicity of zinc ions in the viable epidermis (VE) after dissolution from ZnO NP and penetration into the VE is ill-defined. We therefore quantified the relative concentrations of endogenous and exogenous Zn using a rare stable zinc-67 isotope (67Zn) ZnO NP sunscreen applied to excised human skin and the cytotoxicity of human keratinocytes (HaCaT) using multiphoton microscopy, zinc-selective fluorescent sensing, and a laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) methodology. Multiphoton microscopy with second harmonic generation imaging showed that 67ZnO NPs were retained on the surface or within the superficial layers of the SC. Zn fluorescence sensing revealed higher levels of labile and intracellular zinc in both the SC and VE relative to untreated skin, confirming that dissolved zinc species permeated across the SC into the VE as ionic Zn and significantly not as ZnO NPs. Importantly, the LA-ICP-MS estimated exogenous 67Zn concentrations in the VE of 1.0 ± 0.3 μg/mL are much lower than that estimated for endogenous VE zinc of 4.3 ± 0.7 μg/mL. Furthermore, their combined total zinc concentrations in the VE are much lower than the exogenous zinc concentration of 21 to 31 μg/mL causing VE cytotoxicity, as defined by the half-maximal inhibitory concentration of exogenous 67Zn found in human keratinocytes (HaCaT). This speaks strongly for the safety of ZnO NP sunscreens applied to intact human skin and the associated recent US FDA guidance.

Moving beyond velocity: Opportunities and challenges to quantify immune cell behavior.

The analysis of cellular behavior using intravital multi-photon microscopy has contributed substantially to our understanding of the priming and effector phases of immune responses. Yet, many questions remain unanswered and unexplored. Though advancements in intravital imaging techniques and animal models continue to drive new discoveries, continued improvements in analysis methods are needed to extract detailed information about cellular behavior. Focusing on dendritic cell (DC) and T cell interactions as an exemplar, here we discuss key limitations for intravital imaging studies and review and explore alternative approaches to quantify immune cell behavior. We touch upon current developments in deep learning models, as well as established methods from unrelated fields such as ecology to detect and track objects over time. As developments in open-source software make it possible to process and interactively view larger datasets, the challenge for the field will be to determine how best to combine intravital imaging with multi-parameter imaging of larger tissue regions to discover new facets of leukocyte dynamics and how these contribute to immune responses.

RNO3 QTL regulates vascular structure and arterial stiffness in the spontaneously hypertensive rat.

Increased arterial stiffness is an independent risk factor for hypertension, stroke, and cardiovascular morbidity. Thus, understanding the factors contributing to vascular stiffness is of critical importance. Here, we used a rat model containing a known quantitative trait locus (QTL) on chromosome 3 (RNO3) for vasoreactivity to assess potential genetic elements contributing to blood pressure, arterial stiffness, and their downstream effects on cardiac structure and function. Although no differences were found in blood pressure at any time point between parental spontaneously hypertensive rats (SHRs) and congenic SHR.BN3 rats, the SHRs showed a significant increase in arterial stiffness measured by pulse wave velocity. The degree of arterial stiffness increased with age in the SHRs and was associated with compensatory cardiac changes at 16 wk of age, and decompensatory changes at 32 wk, with no change in cardiac structure or function in the SHR.BN3 hearts at these time points. To evaluate the arterial wall structure, we used multiphoton microscopy to quantify cells and collagen content within the adventitia and media of SHR and SHR.BN3 arteries. No difference in cell numbers or proliferation rates was found, although phenotypic diversity was characterized in vascular smooth muscle cells. Herein, significant anatomical and physiological differences related to arterial structure and cardiovascular tone including collagen, pulse wave velocity (PWV), left ventricular (LV) geometry and function, and vascular smooth muscle cell (VSMC) contractile apparatus proteins were associated with the RNO3 QTL, thus providing a novel platform for studying arterial stiffness. Future studies delimiting the RNO3 QTL could aid in identifying genetic elements responsible for arterial structure and function.

High-Resolution Imaging for the Analysis and Reconstruction of 3D Microenvironments for Regenerative Medicine: An Application-Focused Review.

The rapid evolution of regenerative medicine and its associated scientific fields, such as tissue engineering, has provided great promise for multiple applications where replacement and regeneration of damaged or lost tissue is required. In order to evaluate and optimise the tissue engineering techniques, visualisation of the material of interest is crucial. This includes monitoring of the cellular behaviour, extracellular matrix composition, scaffold structure, and other crucial elements of biomaterials. Non-invasive visualisation of artificial tissues is important at all stages of development and clinical translation. A variety of preclinical and clinical imaging methods—including confocal multiphoton microscopy, optical coherence tomography, magnetic resonance imaging (MRI), and computed tomography (CT)—have been used for the evaluation of artificial tissues. This review attempts to present the imaging methods available to assess the composition and quality of 3D microenvironments, as well as their integration with human tissues once implanted in the human body. The review provides tissue-specific application examples to demonstrate the applicability of such methods on cardiovascular, musculoskeletal, and neural tissue engineering.

Simultaneous scattering compensation at multiple points in multi-photon microscopy.

The two-photon fluorescence imaging depth has been significantly improved in recent years by compensating for tissue scattering with wavefront correction. However, in most approaches the wavefront corrections are valid only over a small sample region on the order of 1 to 10 µm. In samples where most scattering structures are confined to a single plane, sample conjugate correction geometries can increase the observable field to a few tens of µm. Here, we apply a recently introduced fast converging scheme for sensor-less scattering correction termed “Dynamic Adaptive Scattering compensation Holography” (DASH) in a sample conjugate configuration with a high pixel count nematic liquid crystal spatial light modulator (LC-SLM). Using a large SLM allows us to simultaneously correct for scattering at multiple field points, which can be distributed over the entire field of view provided by the objective lens. Despite the comparably slow refresh time of LC-SLMs, we achieve correction times on the order of 10 s per field point, which we show is sufficiently fast to counteract scattering at multiple sites in living mouse hippocampal tissue slices.

Longitudinal Preclinical Imaging Characterizes Extracellular Drug Accumulation After Radiation Therapy in the Healthy and Leukemic Bone Marrow Vascular Microenvironment

Recent initial findings suggest that radiation therapy improves blood perfusion and cellular chemotherapy uptake in mice with leukemia. However, the ability of radiation therapy to influence drug accumulation in the extracellular bone marrow tissue is unknown, due in part to a lack of methodology. This study developed longitudinal quantitative multiphoton microscopy (L-QMPM) to characterize the bone marrow vasculature (BMV) and drug accumulation in the extracellular bone marrow tissue before and after radiation therapy in mice bearing leukemia. We developed a longitudinal window implant for L-QMPM imaging of the calvarium BMV before, 2 days after, and 5 days after total body irradiation (TBI). Live time-lapsed images of a fluorescent drug surrogate were used to obtain measurements, including tissue wash-in slope (WIStissue) to measure extracellular drug accumulation. We performed L-QMPM imaging on healthy C57BL/6 (WT) mice, as well as mice bearing acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Implants had no effects on calvarium dose, and parameters for wild-type untreated mice were stable during imaging. We observed decreased vessel diameter, vessel blood flow, and WIStissue with the onset of AML and ALL. Two to 10 Gy TBI increased WIStissue and vessel diameter 2 days after radiation therapy in all 3 groups of mice and increased single-vessel blood flow in mice bearing ALL and AML. Increased WIStissue was observed 5 days after 10 Gy TBI or 4 Gy split-dose TBI (2 treatments of 2 Gy spaced 3 days apart). L-QMPM provides stable functional assessments of the BMV. Nonmyeloablative and myeloablative TBI increases extracellular drug accumulation in the leukemic bone marrow 2 to 5 days posttreatment, likely through improved blood perfusion and drug exchange from the BMV to the extravascular tissue. Our data show that neo-adjuvant TBI at doses from 2 Gy to 10 Gy conditions the BMV to improve drug transport to the bone marrow.

High energy (>40 nJ), sub-100 fs, 950 nm laser for two-photon microscopy.

Compact and high-energy femtosecond fiber lasers operating around 900-950 nm are desirable for multiphoton microscopy. Here, we demonstrate a >40 nJ, sub-100 fs, wavelength-tunable ultrafast laser system based on chirped pulse amplification (CPA) in thulium-doped fiber and second-harmonic generation (SHG) technology. Through effective control of the nonlinear effect in the CPA process, we have obtained 92-fs pulses at 1903 nm with an average power of 0.89 W and a pulse energy of 81 nJ. By frequency doubling, 95-fs pulses at 954 nm with an average power of 0.46 W and a pulse energy of 42 nJ have been generated. In addition, our system can also achieve tunable wavelength from 932 nm to 962 nm (frequency doubled from 1863 nm to 1919 nm). A pulse width of ∼100 fs and sufficient pulse energy are ensured over the entire tuning range. Finally, we applied the laser in a two-photon microscope and obtained superior imaging results. Due to a relatively low repetition rate (∼ 10 MHz), similar imaging quality can be achieved at significantly reduced average power compared with a commercial 80 MHz laser system. At the same time, the lower average power is helpful in limiting the thermal load to the samples. It is believed that such a setup, with its well-balanced optical characteristics and compact footprint, provides an ideal source for two-photon microscopy.

Estrogen depletion on In vivo osteocyte calcium signaling responses to mechanical loading.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of "osteocyte mechanosomes" - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca2+ channels clustered around αvβ3 integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca2+ indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca2+ signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca2+ signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca2+ signaling osteocytes and loading was unchanged at 2days post-OVX. However, it was altered markedly at 28days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca2+ signaling) osteocytes; however, at higher strains the numbers of Ca2+ signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of αvβ3 foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for αvβ3 and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

A Novel Assay Allowing Drug Self-Administration, Extinction, and Reinstatement Testing in Head-Restrained Mice.

Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.

Experimental determination of shift-less aberration bases for sensorless adaptive optics in nonlinear microscopy.

Adaptive optics can improve the performance of optical systems and devices by correcting phase aberrations. While in most applications wavefront sensing is employed to drive the adaptive optics correction, some microscopy methods may require sensorless optimization of the wavefront. In these cases, the correction is performed by describing the aberration as a linear combination of a base of influence functions, optimizing an image quality metric as a function of the coefficients. The influence functions base is generally chosen to either efficiently represent the adaptive device used or to describe generic wavefronts in an orthogonal fashion. A rarely discussed problem is that most correction bases have elements which introduce, together with a correction of the aberration, a shift of the imaging field of view in three dimensions. While simple methods to solve the problem are available for linear microscopy methods, nonlinear microscopy techniques such as multiphoton or second harmonic generation microscopy require non-trivial base determination. In this paper, we discuss the problem, and we present a method for calibrating a shift-less base on a spatial light modulator for two-photon microscopy.

Time-Lapse Imaging of Root Pathogenesis and Fungal Proliferation Without Physically Disrupting Roots.

Microscopic observation of root disease onset and progression is typically performed by harvesting different plants at multiple time points. This approach prevents the monitoring of individual encounter sites over time, often mechanically damages roots, and exposes roots to unnatural conditions during observation. Here, we describe a method developed to avoid these problems and its application to study Fusarium oxysporum-Arabidopsis thaliana interactions. This method enabled three-dimensional, time-lapse imaging of both A. thaliana and F. oxysporum as they interact via the use of confocal and multi-photon microscopy and facilitated inquiries about the genetic mechanism underpinning Fusarium wilt.

Multiphoton Microscopy for Identifying Collagen Signatures Associated with Biochemical Recurrence in Prostate Cancer Patients.

Prostate cancer is a heterogeneous disease that remains dormant for long periods or acts aggressively with poor clinical outcomes. Identifying aggressive prostate tumor behavior using current glandular-focused histopathological criteria is challenging. Recent evidence has implicated the stroma in modulating prostate tumor behavior and in predicting post-surgical outcomes. However, the emergence of stromal signatures has been limited, due in part to the lack of adoption of imaging modalities for stromal-specific profiling. Herein, label-free multiphoton microscopy (MPM), with its ability to image tissue with stromal-specific contrast, is used to identify prostate stromal features associated with aggressive tumor behavior and clinical outcome. MPM was performed on unstained prostatectomy specimens from 59 patients and on biopsy specimens from 17 patients with known post-surgery recurrence status. MPM-identified collagen content, organization, and morphological tumor signatures were extracted for each patient and screened for association with recurrent disease. Compared to tumors from patients whose disease did not recur, tumors from patients with recurrent disease exhibited higher MPM-identified collagen amount and collagen fiber intensity signal and width. Our study shows an association between MPM-identified stromal collagen features of prostate tumors and post-surgical disease recurrence, suggesting their potential for prostate cancer risk assessment.

Deep Learning-Assisted Multiphoton Microscopy to Reduce Light Exposure and Expedite Imaging in Tissues With High and Low Light Sensitivity.

PurposeTwo-photon excitation fluorescence (2PEF) reveals information about tissue function. Concerns for phototoxicity demand lower light exposure during imaging. Reducing excitation light reduces the quality of the image by limiting fluorescence emission. We applied deep learning (DL) super-resolution techniques to images acquired from low light exposure to yield high-resolution images of retinal and skin tissues.MethodsWe analyzed two methods: a method based on U-Net and a patch-based regression method using paired images of skin (550) and retina (1200), each with low- and high-resolution paired images. The retina dataset was acquired at low and high laser powers from retinal organoids, and the skin dataset was obtained from averaging 7 to 15 frames or 70 frames. Mean squared error (MSE) and the structural similarity index measure (SSIM) were outcome measures for DL algorithm performance.ResultsFor the skin dataset, the patches method achieved a lower MSE (3.768) compared with U-Net (4.032) and a high SSIM (0.824) compared with U-Net (0.783). For the retinal dataset, the patches method achieved an average MSE of 27,611 compared with 146,855 for the U-Net method and an average SSIM of 0.636 compared with 0.607 for the U-Net method. The patches method was slower (303 seconds) than the U-Net method (<1 second).ConclusionsDL can reduce excitation light exposure in 2PEF imaging while preserving image quality metrics.Translational RelevanceDL methods will aid in translating 2PEF imaging from benchtop systems to in vivo imaging of light-sensitive tissues such as the retina.

Label-free imaging of age-related cardiac structural changes in non-human primates using multiphoton nonlinear microscopy.

Heart failure is one of the most common causes of morbidity and mortality. Both maturational abnormalities and age-associated cardiac pathologies contribute to heart failure. Imaging-based assessment to discern detailed cardiac structure at various maturational stages is imperative for understanding mechanisms behind cardiac growth and aging. Using multiphoton nonlinear optical microscopy (NLOM) based label-free imaging, we investigated cardiac structural composition in a human-relevant aging model, the common marmoset monkey (Callithrix jacchus). Animals were divided into three different age groups including neonatal, young adult and old. By devising a unique strategy for segregating collagen and myosin emitted second harmonic generation (SHG) signals, we performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodelling indicating age-related cardiac fibrosis. Significantly low scattering tissue volume in neonatal marmoset hearts was attributed to a lack of binding between the myofibrils in maturing cardiac tissue. Comprehensive quantitative assessment of structural composition during maturation and aging of marmoset hearts revealed significant differences in myofibril length, alignment, curvature and angular distribution. In conclusion, label-free high-resolution NLOM facilitates visualization and quantification of subcellular structural features for understanding vital age-related morphological alterations in the marmoset heart.

Generation of megawatt soliton at 1680 nm in very large mode area antiresonant fiber and application to three-photon microscopy

The spectral window around 1700 nm is interesting for in-depth multiphoton microscopy of intact tissues due to reduced scattering and absorption in this wavelength range. However, wide adoption of this excitation range will rely on the availability of robust and cost-effective high peak power pulsed lasers operating at these wavelengths. Here, we report on a fiber-based femtosecond laser providing up to 95 nJ, 85 fs pulses at 1800 nm. The laser system makes use of a fiber-based chirped pulse amplifier emitting at 1560 nm followed by an in-house fabricated very large mode area antiresonant fiber for soliton self-frequency shift. Megawatt-peak power pulses at the repetition rate of 1 MHz are available directly at the output of the flexible fiber. We illustrate the potential of the source for biological microscopy by recording three-photon-excited fluorescence images of mouse nervous tissue. The flexible fiber tailored to propagate megawatt solitons in the biologically relevant window around 1700 nm opens the way to deep brain imaging of freely moving animals via miniaturized endomicroscopes.