Search

Traumatic penumbra (TP) is a region with recoverable potential around the primary lesion of brain injury. Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury (TBI). In this study, we first established traumatic brain injuries (TBIs) in rats using a modified Feeney method, followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy. The results showed that the technique effectively imaged normal and traumatic brain tissues, and revealed pathological features such as extracellular matrix changes, vascular cell proliferation, and intracellular edema in the traumatic penumbra. Compared with normal brain tissue, the extracellular matrix in the TP was sparse, cells were disorganized, and hyperplastic vascular cells emitted higher two-photon excited fluorescence (TPEF) signals. Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI. [ABSTRACT FROM AUTHOR]

Malignant melanoma is a very aggressive tumour with the ability to metastasize at an early stage. Therefore, early detection is of great importance. Multiphoton tomography is a new non-invasive examination method in the clinical diagnosis of skin alterations that can be used for such early diagnosis. In this paper, a method for automated evaluation of multiphoton images of the skin is presented. The following features at the cellular and subcellular level were extracted to differentiate between malignant melanomas, lesions, and healthy skin: cell symmetry, cell distance, cell density, cell and nucleus contrast, nucleus cell ratio, and homogeneity of cytoplasm. The extracted features formed the basis for the subsequent classification. Two feature sets were used. The first feature set included all the above-mentioned features, while the second feature set included the significantly different features between the three classes resulting from a multivariate analysis of variance. The classification was performed by a Support Vector Machine, the k-Nearest Neighbour algorithm, and Ensemble Learning. The best classification results were obtained with the Support Vector Machine using the first feature set with an accuracy of 52 % and 79.6 % for malignant melanoma and healthy skin, respectively. Despite the small number of subjects investigated our results indicate that the proposed automatic method can differentiate malignant melanoma, lesions, and healthy skin. For future clinical application, an extended study with more multiphoton images is needed.

To investigate the potential mineralising effects of calcium silicate-based dentine replacement material (Biodentine™) in comparison with glass-ionomer cement (GIC) (Fuji IX™) on different human dentine substrates using a multimodal non-invasive optical assessment. Cements were applied on artificially demineralised or naturally carious dentine and stored for 4 weeks in phosphate-rich media +/- tetracycline used for mineralisation labelling. Interfacial dentine was examined from the same sample and location before and after aging using two-photon fluorescence microscopy, fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) imaging. Additionally, Raman spectroscopy was used to detect changes in the mineral content of dentine. Significant changes in the fluorescence intensity and lifetime were detected in partially demineralised dentine and caries-affected dentine underneath both tested cements, after storage (p < 0.001). This was associated with a significant increase in the mineral content as indicated by the increased intensity of the phosphate Raman peak located at 959 cm−1 (p < 0.0001). Caries-infected dentine showed significant fluorescence changes under Biodentine™ after storage (p < 0.001), but not under GIC (p = 0.44). Tetracycline binding induced a reduction in the fluorescence lifetime with comparable increase in the fluorescence intensity in both cements' groups within the affected dentine (p < 0.001). Significance Two-photon fluorescence microscopy can be used efficiently for non-destructive in-vitro dentine caries characterisation providing a technique for studying the same dentine-cement interface over time and detect changes. Biodentine™ demonstrated comparable remineralising potential to GIC, in addition to inducing remineralisation of caries-infected dentine. This may suggest using Biodentine™ as part of minimally invasive operative dentistry (MID) in caries management. [ABSTRACT FROM AUTHOR]

Decision-making in dynamic environments often involves accumulation of evidence, in which new information is used to update beliefs and select future actions. Using in vivo cellular resolution imaging in voluntarily head-restrained rats, we examined the responses of neurons in frontal and parietal cortices during a pulse-based accumulation of evidence task. Neurons exhibited activity that predicted the animal's upcoming choice, previous choice, and graded responses that reflected the strength of the accumulated evidence. The pulsatile nature of the stimuli enabled characterization of the responses of neurons to a single quantum (pulse) of evidence. Across the population, individual neurons displayed extensive heterogeneity in the dynamics of responses to pulses. The diversity of responses was sufficiently rich to form a temporal basis for accumulated evidence estimated from a latent variable model. These results suggest that heterogeneous, often transient sensory responses distributed across the fronto-parietal cortex may support working memory on behavioral timescales. VIDEO ABSTRACT.

Understanding the mechanism of the brain via optical microscopy is one of the challenges in neuroimaging, considering the complex structures. Advanced neuroimaging techniques provide a more comprehensive insight into patho-mechanisms of brain disorders, which is useful in the early diagnosis of the pathological and physiological changes associated with various neurodegenerative diseases. Recent advances in optical microscopy techniques have evolved powerful tools to overcome scattering of light and provide improved in vivo neuroimaging with sub-cellular resolution, endogenous contrast specificity, pinhole less optical sectioning capability, high penetration depth, and so on. The following article reviews the developments in various optical imaging techniques including two-photon and three-photon fluorescence, second-harmonic generation, third-harmonic generation, coherent anti-Stokes Raman scattering, and stimulated Raman scattering in neuroimaging. We have outlined the potentials and drawbacks of these techniques and their possible applications in the investigation of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Objective: To utilize Multiphoton Microscopy (MPM) as a novel imaging technique to characterize and quantify collagen at the Renal Cell Carcinoma Pseudocapsule, to assess for both intra-tumoral and inter-tumoral variation of collagen characteristics. MPM combines Second Harmonic Generation and Two Photon Excitation Fluorescence to image extracellular matrix architecture.Methods: Twenty partial nephrectomy specimen tissues were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The pseudocapsules (PCs) were imaged with 2X and 20X objective at selected Regions of Interest. Corresponding clinical information was retrieved. PC thickness was determined. Collagen parameters measured included quantification by the Collagen Area Ratio, and qualitative measurements by the Collagen Fiber Density and Collagen Reticulation Index.Results: The boundaries between tumor, PC and normal renal parenchyma were distinguished by MPM without need for staining. In the thickest areas of the PC, collagen content and density were quantitatively higher compared to the thinnest areas. Median Collagen Area Ratio was higher in the thickest compared to the thinnest areas of the PC (P = .01). Clear Cell RCC specimens had a consistently higher Collagen Fiber Density in both the thickest and thinnest areas compared to non-Clear Cell RCC specimens (P = .02).Conclusions: We demonstrated the ability of MPM to quantify collagen characteristics of PCs without fluorescent labeling. Tumor enucleation for Renal Cell Carcinoma along its PC remains debatable with regards to oncological safety. Even with a complete and intact PC, the PC is not a homogenous structure, and varies in its thickness and its collagen characteristics within, and between tumors. [ABSTRACT FROM AUTHOR]

Objectives: The investigation of cochlear hair cells and lateral wall is a time‐consuming and labor‐intensive process. However, it is a mandatory experiment in audiology research. Here we suggest a novel method for investigating the inner ear microstructures from intact cochleae using two‐photon laser scanning microscopy (TPLSM). This technique guarantees fewer artifacts and technical simplicity. Methods: Using TPLSM, we investigated the whole mount cochleae, decalcified cochleae, and cleared cochleae of wild type C57BL/6 mice. CX3CR1+/GFP mice were used to investigate the feasibility of visualizing cellular structures in the cochlear spiral ligament. All samples were investigated without staining. Results: Endogenous fluorescence emission from the outer hair cells was strong enough to be distinguished from the other structures in all samples. From the single apical view, 50 and 90% of the whole hair cells of the decalcified cochleae and cleared cochleae, respectively, could be visualized without staining using TPLSM. Capillary structure of stria vascularis and spiral ligament could be visualized by endogenous fluorescence without staining. Conclusion: We successfully investigated the hair cells and lateral wall of mouse cochleae using TPLSM without using staining or any destructive procedures. This method is easier, faster, and more reliable than conventional methods. [ABSTRACT FROM AUTHOR]

Background: Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and antiplatelet therapeutics are combined. Prior studies have shown different effects of antiplatelet vs anticoagulant drugs on the structure and function of hemostatic plugs in vivo., Objectives: We examined whether dual antithrombotic treatment consisting of combined antiplatelet and anticoagulant therapeutics alters hemostatic plug structure and function differently from treatment with either therapeutic alone., Methods: Mice were treated with the P2Y 12 antagonist clopidogrel and the factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy, and bleeding times were recorded., Results: Mice treated with clopidogrel alone exhibited a decrease in platelet accumulation at the site of injury, with prolonged bleeding times only at the highest doses of clopidogrel used. Mice treated with rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban exhibited platelet and fibrin accumulation that was similar to that with either drug given alone; however, dual antithrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation., Conclusion: Combined administration of antiplatelet and anticoagulant therapeutics exacerbates bleeding as compared to that with either drug alone, potentially via combined loss of both adenosine 5'-diphosphate- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

In the postnatal mammalian brain, neural stem cells of the ventricular–subventricular zone continue to generate doublecortin (Dcx)‐expressing immature neurons. Throughout life, these immature neurons migrate to the olfactory bulb through the rostral migratory stream (RMS). In this study, we investigated the distribution of these putative immature neurons using enhanced green fluorescent protein (EGFP) expression in the area surrounding the RMS of the juvenile Dcx‐EGFP mice. Through the combined use of an optical clearing reagent (a 2,2′‐thiodiethanol solution) and two‐photon microscopy, we visualized three‐dimensionally the EGFP‐positive cells in the entire RMS and its surroundings. The resulting wide‐field and high‐definition images along with computational image processing methods developed in this study were used to comprehensively determine the position of the EGFP‐positive cells. Our findings revealed that the EGFP‐positive cells were heterogeneously distributed in the area surrounding the RMS. In addition, the orientation patterns of the leading process of these cells, which displayed the morphology of migrating immature neurons, differed depending on their location. These novel results provide highly precise morphological information for immature neurons and suggest that a portion of immature neurons may be detached from the RMS and migrate in various directions. Through a combination of 3D imaging techniques and the computational image processing protocols, we revealed that numerous Dcx‐expressing cells were heterogeneously distributed in the areas surrounding the rostral migratory stream (RMS) in the juvenile mouse. The orientation of leading process of Dcx‐expressing cells differed depending on their locations. [ABSTRACT FROM AUTHOR]

Urodynamic tests are the gold standard for the diagnosis of bladder dysfunction, and the mechanical compliance of the bladder is an important parameter in these tests. The bladder wall has a layered structure, differentially affected by pathology, so knowledge of the contribution and role of these layers and their constituents to overall bladder compliance will enhance interpretation of these clinical tests. In this study we document the functional morphology of the detrusor and lamina propria of the murine bladder wall using a custom in-situ tensile loading system under multiphoton microscopy (MPM) observation in unloaded state and under incremental uniaxial stretch. Features in the stress-stretch curves of bladder samples were then directly related to corresponding MPM images. Collagen organisation across wall depth was quantified using image analysis techniques. The hypothesis that the lamina propria deformed at low strain by unfolding of the rugae and rearranging collagen fibrils was confirmed. A novel ‘pocket’ feature in the detrusor was observed along with extensive rearrangement of fibrils in two families at different depths, providing higher stiffness at high stretches in the detrusor. The very different deformations of detrusor and lamina propria were accommodated by the highly coiled structure of collagen in the lamina propria. Imaging and mechanical studies presented here allow gross mechanical response to be attributed to specific components of the bladder wall and further, may be used to investigate the impact of microstructural changes due to pathology or aging, and how they impair tissue functionality. Statement of Significance This article reports the first in-situ multiphoton microscopy observations of microstructural deformation under uniaxial tensile loading of ex vivo bladder. We describe collagen rearrangement through the tissue thickness and relate this directly to the stress-stretch behaviour. We confirm for the first time the unfolding of rugae and realignment of fibrils in the lamina propria during extension and the rapid stiffening as two fibril families in the detrusor are engaged. This technique provides new insight into microstructure function and will enhance understanding of the impact of changes due to pathology or aging. [ABSTRACT FROM AUTHOR]

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells' shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes. [ABSTRACT FROM AUTHOR]

Thymocytes migrate through discrete compartments within the thymus, engaging in cellular interactions essential for their differentiation into functional and self-tolerant T cells. Thus, understanding the temporal and spatial behavior of thymocytes within an intact thymic microenvironment is critical for elucidating processes governing T cell development. Towards this end, we describe methods for preparing thymic explant slices, in which the migration of thymocytes through three-dimensional space can be probed using time-lapse, multiphoton fluorescence microscopy. Thymocytes, enriched for developmental subsets of interest, are labeled with cytoplasmic fluorescent dyes, and seeded onto live thymic slices that express an endogenous, stromal cell-specific fluorescent reporter. In response to chemotactic cues produced by thymic stromal cells, the labeled thymocytes migrate withinthymic microenvironments and engage in cellular interactions that recapitulate a physiological system, whichcan be readily imaged. Here we describe specimen preparation that maintains the integrity of thymic structures. We also describe imaging protocols for acquiring multiple fluorochrome channels to enable detection of thymocyte:stromal cell interactions and quantification of relative intracellular calcium levels to monitor T cell receptor activation. Parameters for quantifying motility and interaction behaviors during data analysis are also briefly described. The thymic slice is a versatile tool for probing live cell behaviors and developing novel hypotheses not readily apparent by static experimental methods.