Two-Photon and Multiphoton Microscopy in Anterior Segment Diseases of the Eye.

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

Role of in vivo imaging in Head and Neck cancer management.

Intravital microscopy (IVM) and optical coherency tomography (OCT) are powerful optical imaging tools that allow visualization of dynamic biological activities in living subjects with subcellular resolutions. They have been used in preclinical and clinical cancer imaging, providing insights into the complex physiological, cellular, and molecular behaviors of tumors. They have revolutionized cancer diagnosis and therapies, allowing for real-time observation of biologic processes in vivo, including angiogenesis and immune cell interactions. Recent developments in techniques for observing deep tissues of living animals have improved bioluminescent proteins, fluorescent proteins, fluorescent dyes, and detection technologies like two-photon excitation microscopy. These technologies have become indispensable tools in basic sciences, preclinical research, and modern drug development. In Vivo imaging can detect subcellular signaling or metabolic events in living animals, but depth-dependent signal attenuation limits the depth from which significant data can be obtained. Cancer cell motility and invasion are key features of metastatic tumors, but only a small portion of tumor cells are motile and metastasize due to genetic, epigenetic, and microenvironmental heterogeneities.

Region- and layer-specific investigations of the human menisci using SHG imaging and biaxial testing.

In this paper, we examine the region- and layer-specific collagen fiber morphology via second harmonic generation (SHG) in combination with planar biaxial tension testing to suggest a structure-based constitutive model for the human meniscal tissue. Five lateral and four medial menisci were utilized, with samples excised across the thickness from the anterior, mid-body, and posterior regions of each meniscus. An optical clearing protocol enhanced the scan depth. SHG imaging revealed that the top samples consisted of randomly oriented fibers with a mean fiber orientation of 43.3 o . The bottom samples were dominated by circumferentially organized fibers, with a mean orientation of 9.5 o . Biaxial testing revealed a clear anisotropic response, with the circumferential direction being stiffer than the radial direction. The bottom samples from the anterior region of the medial menisci exhibited higher circumferential elastic modulus with a mean value of 21 MPa. The data from the two testing protocols were combined to characterize the tissue with an anisotropic hyperelastic material model based on the generalized structure tensor approach. The model showed good agreement in representing the material anisotropy with a mean r 2 = 0.92.

Value of multiphoton microscopy in uro-oncology: a narrative review.

Background and Objective: Multi-photon microscopy (MPM) is a 3-dimension fluorescence imaging technique that combines the excitation of two low-energy photons, enabling less photo-bleaching and deeper penetration of the imaged tissue. Two signals are detected, autofluorescence (AF), from natural intracellular fluorophores [such as nicotinamide adenine dinucleotide phosphate (NADP) and flavine adenine dinucleotide (FAD) transformation], and second harmonic generation (SHG), a physical property of the laser enhancing non-centrosymmetric structures such as collagen fibers. MPM can give both visual and quantitative information of a fresh tissue (without the need of processing, cutting or staining the tissue), aiding in the progress towards optimizing a real-time imaging device. The objective of this review is to show the value and benefits of the use of MPM in uro-oncology. Methods: A structured literature review was performed using PubMed and Web of Sciences, including all articles with the following keywords: "multiphoton microscopy", "two-photon microscopy", "non-linear microscopy", "second harmonic generation", "urology", "prostate", "bladder", "kidney", "upper tract", "oncology", "surgical margins", "frozen section". Articles were reviewed to summarize the use of this tool in performing biopsies, assessing surgical margins, staging and grading complementary tool, and real-time imaging. Key Content and Findings: A total of 476 articles were identified with these keywords, and later screened for inclusion. We finally included 47 publications that were relevant to our topic. The advantages of this technique have led to its application in the management of several cancers, allowing cellular description as well as quantitative measurements of AF or SHG and their correlation with clinical outcomes. Conclusions: MPM has shown great improvement in providing a real time assessment of fresh tissue, giving oncologic diagnosis, performing in vivo imaging and quantitative analysis of the tissue as well as increasing precision of the diagnosis. This nonlinear optical technique has the potential of guiding both biopsy and surgery, as well as helping the surgeon with interesting additional tissue information intra-operatively.

Changes in the microstructure of the human aortic adventitia under biaxial loading investigated by multi-photon microscopy.

Among the three layers of the aortic wall, the media is primarily responsible for its mechanical properties, but the adventitia prevents the aorta from overstretching and rupturing. The role of the adventitia is therefore crucial with regard to aortic wall failure, and understanding the load-induced changes in tissue microstructure is of high importance. Specifically, the focus of this study is on the changes in collagen and elastin microstructure in response to macroscopic equibiaxial loading applied to the aortic adventitia. To observe these changes, multi-photon microscopy imaging and biaxial extension tests were performed simultaneously. In particular, microscopy images were recorded at 0.02 stretch intervals. The microstructural changes of collagen fiber bundles and elastin fibers were quantified with the parameters of orientation, dispersion, diameter, and waviness. The results showed that the adventitial collagen was divided from one into two fiber families under equibiaxial loading conditions. The almost diagonal orientation of the adventitial collagen fiber bundles remained unchanged, but the dispersion was substantially reduced. No clear orientation of the adventitial elastin fibers was observed at any stretch level. The waviness of the adventitial collagen fiber bundles decreased under stretch, but the adventitial elastin fibers showed no change. These original findings highlight differences between the medial and adventitial layers and provide insight into the stretching process of the aortic wall. STATEMENT OF SIGNIFICANCE: To provide accurate and reliable material models, it is essential to understand the mechanical behavior of the material and its microstructure. Such understanding can be enhanced with tracking of the microstructural changes caused by mechanical loading of the tissue. This study provides therefore a unique dataset of structural parameters of the human aortic adventitia obtained under equibiaxial loading. The structural parameters describe orientation, dispersion, diameter, and waviness of collagen fiber bundles and elastin fibers. Eventually, the microstructural changes in the human aortic adventitia are compared with the microstructural changes in the human aortic media from a previous study. This comparison reveals the cutting-edge findings on the differences in the response to the loading between these two human aortic layers.

Multi-Photon Microscopy.

In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse. © 2023 Wiley Periodicals LLC.

SpecSeg is a versatile toolbox that segments neurons and neurites in chronic calcium imaging datasets based on low-frequency cross-spectral power.

Imaging calcium signals in neurons of animals using single- or multi-photon microscopy facilitates the study of coding in large neural populations. Such experiments produce massive datasets requiring powerful methods to extract responses from hundreds of neurons. We present SpecSeg, an open-source toolbox for (1) segmentation of regions of interest (ROIs) representing neuronal structures, (2) inspection and manual editing of ROIs, (3) neuropil correction and signal extraction, and (4) matching of ROIs in sequential recordings. ROI segmentation in SpecSeg is based on temporal cross-correlations of low-frequency components derived by Fourier analysis of each pixel with its neighbors. The approach is user-friendly, intuitive, and insightful and enables ROI detection around neurons or neurites. It works for single- (miniscope) and multi-photon microscopy data, eliminating the need for separate toolboxes. SpecSeg thus provides an efficient and versatile approach for analyzing calcium responses in neuronal structures imaged over prolonged periods of time.

Changes in the microstructure of the human aortic medial layer under biaxial loading investigated by multi-photon microscopy.

Understanding the correlation between tissue architecture, health status, and mechanical properties is essential for improving material models and developing tissue engineering scaffolds. Since structural-based material models are state of the art, there is an urgent need for experimentally obtained structural parameters. For this purpose, the medial layer of nine human abdominal aortas was simultaneously subjected to equibiaxial loading and multi-photon microscopy. At each loading interval of 0.02, collagen and elastin fibers were imaged based on their second-harmonic generation signal and two-photon excited autofluorescence, respectively. The structural alterations in the fibers were quantified using the parameters of orientation, diameter, and waviness. The results of the mechanical tests divided the sample cohort into the ruptured and non-ruptured, and stiff and non-stiff groups, which were covered by the findings from histological investigations. The alterations in structural parameters provided an explanation for the observed mechanical behavior. In addition, the waviness parameters of both collagen and elastin fibers showed the potential to serve as indicators of tissue strength. The data provided address deficiencies in current material models and bridge multiscale mechanisms in the aortic media. STATEMENT OF SIGNIFICANCE: Available material models can reproduce, but cannot predict, the mechanical behavior of human aortas. This deficiency could be overcome with the help of experimentally validated structural parameters as provided in this study. Simultaneous multi-photon microscopy and biaxial extension testing revealed the microstructure of human aortic media at different stretch levels. Changes in the arrangement of collagen and elastin fibers were quantified using structural parameters such as orientation, diameter and waviness. For the first time, structural parameters of human aortic tissue under continuous loading conditions have been obtained. In particular, the waviness parameters at the reference configuration have been associated with tissue stiffness, brittleness, and the onset of atherosclerosis.

Linking the region-specific tissue microstructure to the biaxial mechanical properties of the porcine left anterior descending artery.

Coronary atherosclerosis is the main cause of death worldwide. Advancing the understanding of coronary microstructure-based mechanics is fundamental for the development of therapeutic tools and surgical procedures. Although the passive biaxial properties of the coronary arteries have been extensively explored, their regional differences and the relationship between tissue microstructure and mechanics have not been fully characterized. In this study, we characterized the passive biaxial mechanical properties and microstructural properties of the proximal, medial, and distal regions of the porcine left anterior descending artery (LADA). We also attempted to relate the biaxial stress-stretch response of the LADA and its respective birefringent responses to the polarized light for obtaining information about the load-dependent microstructural variations. We found that the LADA extensibility is reduced in the proximal-to-distal direction and that the medial region exhibits more heterogeneous mechanical behavior than the other two regions. We have also observed highly dynamic microstructural behavior where fiber families realign themselves depending on loading. In addition, we found that the microstructure of the distal region exhibited highly aligned fibers along the longitudinal axis of the artery. To verify this microstructural feature, we imaged the LADA specimens with multi-photon microscopy and observed that the adventitia microstructure transitioned from a random fiber network in the proximal region to highly aligned fibers in the distal region. Our findings could offer new perspectives for understanding coronary mechanics and aid in the development of tissue-engineered vascular grafts, which are currently limited due to their mismatch with native tissue in terms of mechanical properties and microstructural features. STATEMENT OF SIGNIFICANCE: The tissue biomechanics of coronary arteries is fundamental for the development of revascularization techniques such as coronary artery bypass. These therapeutics require a deep understanding of arterial mechanics, microstructure, and mechanobiology to prevent graft failure and reoperation. The present study characterizes the unique regional mechanical and microstructural properties of the porcine left anterior descending artery using biaxial testing, polarized-light imaging, and confocal microscopy. This comprehensive characterization provides an improved understanding of the collagen/elastin architecture in response to mechanical loads using a region-specific approach. The unique tissue properties obtained from this study will provide guidance for the selection of anastomotic sites in coronary artery bypass grafting and for the design of tissue-engineered vascular grafts.

Multi-photon microscopy for the evaluation of interstitial fibrosis in extended criteria donor kidneys: A proof-of-concept study.

INTRODUCTION: To evaluate the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence in multiphoton microscopy (MPM) for the quantification of collagen/fibrosis on preimplantation biopsies of extended criteria donors (ECD). MATERIALS AND METHODS: Twenty preimplantation core biopsies were extracted from 10 donor kidney samples, of which originated from seven donors. Kidney Donor Profile Index (KDPI) and Remuzzi scores of biopsies were calculated. Collagen parameters measured included quantification by the Collagen Area Ratio in Total Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI). RESULTS: Biopsies classified with > 85% KDPI scores had significantly higher CART (p = .011) and lower CRI values (p = .025) than biopsies with ≤ 85% KDPI scores. Increase in CRI values correlated significantly with rise in recipient creatinine levels 1-year post-transplant (p = .027; 95% CI: 4.635-66.797). CONCLUSION: MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained preimplantation biopsies of donor kidneys stratified by KDPI scores. This initial evaluation found significant differences in both parameters between donor kidneys with more or less than 85% KDPI.

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging.

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.

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.

Hot-Band-Absorption-Induced Anti-Stokes Fluorescence of Aggregation-Induced Emission Dots and the Influence on the Nonlinear Optical Effect.

Hot-band absorption (HBA)-induced anti-Stokes fluorescence (ASF) with longer-wavelength excitation is one effective pathway to deep penetration and low autofluorescence in intravital fluorescence imaging, raising demands for fluorophores with broad spectra, high absorption, and strong emission. However, typical fluorescent dyes display some emission quenching when their concentration is increased in order to obtain brighter fluorescence. In this work, the HBA-induced ASF of aggregation-induced emission (AIE) dots is reported. BPN-BBTD dots were synthesized and confirmed with a fluorescence enhancement and a considerable ASF intensity. In addition, the mechanism of ASF and the HBA process of BPN-BBTD dots were carefully validated and discussed. To obtain the full advantages of the long-wavelength excitation and the short fluorescence lifetime in deep-tissue bioimaging, a large-depth ASF confocal microscopic imaging of in vivo cerebral vasculature was conducted under the excitation of a 980 nm continuous wave laser after intravenous injection of BPN-BBTD dots. Meanwhile, the 3D structure of the cerebrovascular network was successfully reconstructed.

Role of Pial Microvasospasms and Leukocyte Plugging for Parenchymal Perfusion after Subarachnoid Hemorrhage Assessed by In Vivo Multi-Photon Microscopy.

Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia. We suggested spasms of pial arterioles as a possible mechanism; however, it remained unclear whether and how pial microvasospasms (MVSs) induce cerebral ischemia. Therefore, we used in vivo deep tissue imaging by two-photon microscopy to investigate MVSs together with the intraparenchymal microcirculation in a clinically relevant murine SAH model. Male C57BL/6 mice received a cranial window. Cerebral vessels and leukocytes were labelled with fluorescent dyes and imaged by in vivo two-photon microscopy before and three hours after SAH induced by filament perforation. After SAH, a large clot formed around the perforation site at the skull base, and blood distributed along the perivascular space of the middle cerebral artery up to the cerebral cortex. Comparing the cerebral microvasculature before and after SAH, we identified three different patterns of constrictions: pearl string, global, and bottleneck. At the same time, the volume of perfused intraparenchymal vessels and blood flow velocity in individual arterioles were significantly reduced by more than 60%. Plugging of capillaries by leukocytes was observed but infrequent. The current study demonstrates that perivascular blood is associated with spasms of pial arterioles and that these spasms result in a significant reduction in cortical perfusion after SAH. Thus, the pial microvasospasm seems to be an important mechanism by which blood in the subarachnoid space triggers cerebral ischemia after SAH. Identifying the mechanisms of pial vasospasm may therefore result in novel therapeutic options for SAH patients.

A quantitative analysis of bone lamellarity and bone collagen linearity induced by distinct dosing and frequencies of teriparatide administration in ovariectomized rats and monkeys.

The lamellar structure of bone, which endows biomechanical rigidity to support the host organism, is observed in mammals, including humans. It is therefore essential to develop a quantitative analysis to evaluate the lamellarity of bone, which would especially be useful for the pharmacological evaluation of anti-osteoporotic drugs. This study applied a current system for the semi-automatic recognition of fluorescence signals to the analysis of un-decalcified bone sections from rat and monkey specimens treated with teriparatide (TPTD). Our analyses on bone formation pattern and collagen topology indicated that TPTD augmented bone lamellarity and bone collagen linearity, which were possibly associated with the recovery of collagen cross-linking, thus endowing bone rigidity.

Tissue Imaging and Quantification Relying on Endogenous Contrast.

Cell-matrix interactions play an important role in regulating a variety of essential processes in multicellular organisms, and are closely associated with numerous diseases. Modified interactions have major effects upon key features of both cells and extracellular matrix (ECM), and a thorough understanding of changes in these features can lead to critically important insights of diseases as well as the identification of effective therapeutic targets. Here, we summarize recent advances in quantitative, optical imaging of cellular metabolism and ECM spatial organization using endogenous sources of contrast. Specifically, we focus on the two-photon excited fluorescence (TPEF) imaging of autofluorescent cellular coenzymes, NAD(P)H and FAD, for the extraction of metabolic information described by optical biomarkers including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial clustering. We show representative applications in assessing adipose tissue function and detecting malignant lesions in human skin, and further demonstrate that a combination of these optical metrics can provide complementary insights into the underlying biological mechanisms. In addition, we review the development of quantitative analysis methods to extract spatial orientation and organization metrics of collagen fibers, a major ECM component, and demonstrate applications of these approaches in two and three dimensions in several diseases, including would healing, osteoarthritis and cancer, as well as assessments of matrix remodeling in hormone-regulated engineered breast tissues. Finally, we summarize this chapter and discuss important research directions that we expect will evolve in the near future.

Spatio-temporal parameters for optical probing of neuronal activity.

The challenge to understand the complex neuronal circuit functions in the mammalian brain has brought about a revolution in light-based neurotechnologies and optogenetic tools. However, while recent seminal works have shown excellent insights on the processing of basic functions such as sensory perception, memory, and navigation, understanding more complex brain functions is still unattainable with current technologies. We are just scratching the surface, both literally and figuratively. Yet, the path towards fully understanding the brain is not totally uncertain. Recent rapid technological advancements have allowed us to analyze the processing of signals within dendritic arborizations of single neurons and within neuronal circuits. Understanding the circuit dynamics in the brain requires a good appreciation of the spatial and temporal properties of neuronal activity. Here, we assess the spatio-temporal parameters of neuronal responses and match them with suitable light-based neurotechnologies as well as photochemical and optogenetic tools. We focus on the spatial range that includes dendrites and certain brain regions (e.g., cortex and hippocampus) that constitute neuronal circuits. We also review some temporal characteristics of some proteins and ion channels responsible for certain neuronal functions. With the aid of the photochemical and optogenetic markers, we can use light to visualize the circuit dynamics of a functioning brain. The challenge to understand how the brain works continue to excite scientists as research questions begin to link macroscopic and microscopic units of brain circuits.

4D Live Imaging and Analysis of Chick Embryo Somites.

Avian (chick) embryos are an established and accessible model organism making them ideal for studying developmental processes. Chick embryos can be harvested from the egg and cultured allowing real-time observations and imaging. Here, we describe ex vivo culture and preparation of somite tissue followed by time-lapse multi-photon microscopy, image capture and processing. We applied this approach to perform live imaging of somites, the paired segments in vertebrate embryos that form in a regular sequence on either side of the neural tube, posteriorly from presomitic mesoderm (psm). Somites give rise to cell lineages of the musculoskeletal system in the trunk such as skeletal muscle, cartilage and tendon, as well as endothelial cells. Until recently it was not possible to observe the cellular dynamics underlying morphological transitions in live tissue, including in somites which undergo epithelial-to-mesenchymal transitions (EMT) during their differentiation. In addition to the experimental setup, we describe the analytical tools used for image processing.

Imaging of muscle activity-induced morphometric changes in fibril network of myofascia by two-photon microscopy.

Myofascia, deep fascia enveloping skeletal muscles, consists of abundant collagen and elastin fibres that play a key role in the transmission of muscular forces. However, understanding of biomechanical dynamics in myofascia remains very limited due to less quantitative and relevant approaches for in vivo examination. The purpose of this study was to evaluate the myofascial fibril structure by means of a quantitative approach using two-photon microscopy (TPM) imaging in combination with intravital staining of Evans blue dye (EBD), a far-red fluorescence dye, which potentially labels elastin. With focus on myofascia of the tibial anterior (TA) muscle, the fibril structure intravitally stained with EBD was observed at the depth level of collagen fibrous membrane above the muscle belly. The EBD-labelled fibril structure and orientation in myofascia indicated biomechanical responses to muscle activity and ageing. The orientation histograms of EBD-labelled fibrils were significantly modified depending upon the intensity of muscle activity and ageing. Moreover, the density of EBD-labelled fibrils in myofascia decreased with habitual exercise but increased with muscle immobilization or ageing. In particular, the diameter of EBD-labelled fibrils in aged mice was significantly higher. The orientation histograms of EBD-labelled fibrils after habitual exercise, muscle immobilization and ageing showed significant differences compared to control. Indeed, the histograms in bilateral TA myofascia of exercise mice made simple waveforms without multiple sharp peaks, whilst muscular immobilization or ageing significantly shifted a histogram with sustaining multiple sharp peaks. Therefore, the dynamics of fibre network with EBD fluorescence in response to the biomechanical environment possibly indicate functional tissue adaptation in myofascia. Furthermore, on the basis of the knowledge that neutrophil recruitment occurs locally in working muscles, we suggested the unique reconstruction mechanism involving neutrophilic elastase in the myofascial fibril structure. In addition to the elastolytic susceptibility of EBD-labelled fibrils, distinct immunoreactivities and activities of neutrophil elastase in the myofascia were observed after electric pulse stimulation-induced muscle contraction for 15 min. Our findings of EBD-labelled fibril dynamics in myofascia through quantitative approach using TPM imaging and intravital fluorescence labelling potentially brings new insights to examine muscle physiology and pathology.

The sarcomere force-length relationship in an intact muscle-tendon unit.

The periodic striation pattern in skeletal muscle reflects the length of the basic contractile unit: the sarcomere. More than half a century ago, Gordon, Huxley and Julian provided strong support for the 'sliding filament' theory through experiments with single muscle fibres. The sarcomere force-length (FL) relationship has since been extrapolated to whole muscles in an attempt to unravel in vivo muscle function. However, these extrapolations were frequently associated with non-trivial assumptions, such as muscle length changes corresponding linearly to SL changes. Here, we determined the in situ sarcomere FL relationship in a whole muscle preparation by simultaneously measuring muscle force and individual SLs in an intact muscle-tendon unit (MTU) using state-of-the-art multi-photon excitation microscopy. We found that despite great SL non-uniformity, the mean value of SLs measured from a minute volume of the mid-belly, equivalent to about 5×10-6% of the total muscle volume, agrees well with the theoretically predicted FL relationship, but only if the precise contractile filament lengths are known, and if passive forces from parallel elastic components and activation-associated sarcomere shortening are considered properly. As SLs are not uniformly distributed across the whole muscle and changes in SL with muscle length are location dependent, our results may not be valid for the proximal or distal parts of the muscle. The approach described here, and our findings, may encourage future studies to determine the role of SL non-uniformity in influencing sarcomere FL properties in different muscles and for different locations within single muscles.