AutoLNMNet: Automated Network for Estimating Lymph-Node Metastasis in EGC Using a Pyramid Vision Transformer and Data Derived From Multiphoton Microscopy.

Lymph-node status is important in decision-making during early gastric cancer (EGC) treatment. Currently, endoscopic submucosal dissection is the mainstream treatment for EGC. However, it is challenging for even experienced endoscopists to accurately diagnose and treat EGC. Multiphoton microscopy can extract the morphological features of collagen fibers from tissues. The characteristics of collagen fibers can be used to assess the lymph-node metastasis status in patients with EGC. First, we compared the accuracy of four deep learning models (VGG16, ResNet34, MobileNetV2, and PVTv2) in training preprocessed images and test datasets. Next, we integrated the features of the best-performing model, which was PVTv2, with manual and clinical features to develop a novel model called AutoLNMNet. The prediction accuracy of AutoLNMNet for the no metastasis (Ly0) and metastasis in lymph nodes (Ly1) stages reached 0.92, which was 0.3% higher than that of PVTv2. The receiver operating characteristics of AutoLNMNet in quantifying Ly0 and Ly1 stages were 0.97 and 0.97, respectively. Therefore, AutoLNMNet is highly reliable and accurate in detecting lymph-node metastasis, providing an important tool for the early diagnosis and treatment of EGC.

Near-Infrared Emissive π-Conjugated Oligomer Nanoparticles for Three- and Four-Photon Deep-Brain Microscopic Imaging Beyond 1700 nm Excitation.

High-resolution visualization of the deep brain is still a challenging and very significant issue. Multiphoton microscopy (MPM) holds great promise for high-spatiotemporal deep-tissue imaging under NIR-III and NIR-IV excitation. However, thus far, their applications have been seriously restricted by the scarcity of efficient organic probes. Herein, we designed and synthesized two donor-acceptor-donor-type conjugated small molecules (TNT and TNS) for in vivo mouse deep-brain imaging with three- and four-photon microscopy under 1700 and 2200 nm excitation. With a selenium (Se) substitution, we synthesized two conjugated small molecules to promote their emission into the deep near-infrared region with high quantum yields of 55% and 20% in THF solvent, respectively, and their water-dispersive nanoparticles have relatively large absorption cross-sections in the 1700 and 2200 nm windows, respectively, with good biosafety. With these superiorities, these organic NPs achieve high-resolution deep-brain imaging via three-photon and four-photon microscopy with excitation at 1700 and 2200 nm windows, and 1620 µm deep in the brain vasculature can be visualized in vivo. This study demonstrates the efficiency of NIR-emissive conjugated small molecules for high-performance MPM imaging in the NIR-III and NIR-IV window and provides a route for the future design of organic MPM probes.

Ultralow-dose irradiation enables engraftment and intravital tracking of disease initiating niches in clonal hematopoiesis.

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion, governing progression, and leukemic transformation of hematological clonal disorders. However, our ability to interrogate the niche in pre-malignant conditions has been limited, as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here, we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity, cell viability, and HSC-supportive functions of the stromal cells, suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2+/- and its healthy counterpart, showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably, stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context.

Fast Multiphoton Microscopic Imaging Joint Image Super-Resolution for Automated Gleason Grading of Prostate Cancers.

Gleason grading system is dependable for quantifying prostate cancer. This paper introduces a fast multiphoton microscopic imaging method via deep learning for automatic Gleason grading. Due to the contradiction between multiphoton microscopy (MPM) imaging speed and quality, a deep learning architecture (SwinIR) is used for image super-resolution to address this issue. The quality of low-resolution image is improved, which increased the acquisition speed from 7.55 s per frame to 0.24 s per frame. A classification network (Swin Transformer) was introduced for automated Gleason grading. The classification accuracy and Macro-F1 achieved by training on high-resolution images are respectively 90.9% and 90.9%. For training on super-resolution images, the classification accuracy and Macro-F1 are respectively 89.9% and 89.9%. It shows that super-resolution image can provide a comparable performance to high-resolution image. Our results suggested that MPM joint image super-resolution and automatic classification methods hold the potential to be a real-time clinical diagnostic tool for prostate cancer diagnosis.

Bone turnover markers in the preoperative assessment of bone quality - A prospective investigation of bone microstructure and advanced glycation endproducts in lumbar fusion patients.

INTRODUCTION: Effective tools to evaluate bone quality preoperatively are scarce and the standard method to determine bone quality requires an invasive biopsy. A non-invasive, and preoperatively available method for bone quality assessment would be of clinical value. The purpose of this study is to investigate the associations of bone formation marker, serum bone alkaline phosphatase (BAP), and bone resorption marker, urine collagen cross-linked N-telopeptide (uNTX) to volumetric bone mineral density (vBMD), fluorescent advanced glycation endproducts (fAGEs) and bone microstructure. MATERIALS AND METHODS: A cross-secional analysis using prospective data of patients undergoing lumbar spinal fusion was performed. BAP and uNTX were preoperatively collected. Quantitative computed tomography (QCT) was performed at the lumbar spine (vBMD ≤ 120 mg/cm3 osteopenic/osteoporotic). Bone biopsies from the posterior superior iliac spine were obtained and evaluated with multiphoton fluorescence microscopy for fAGEs and microcomputed tomography (µCT) for bone microarchitecture. Correlations between BAP/uNTX to vBMD, fAGEs and µCT parameters were assessed with Spearman's ρ. Receiver operating characteristic (ROC) analysis evaluated BAP and uNTX as predictors for osteopenia/osteoporosis. Multivariable linear regression models adjusting for age, sex, BMI, race and diabetes mellitus determined associations between BAP/uNTX and fAGEs. RESULTS: 127 prospectively enrolled patients (50.4% female, 62.5 years, BMI 28.7 kg/m2) were analyzed. uNTX (ρ=-0.331,p < 0.005) and BAP (ρ=-0.245,p < 0.025) decreased with cortical fAGEs, and uNTX (ρ=-0.380,p < 0.001) decreased with trabecular fAGEs. BAP and uNTX revealed no significant correlation with vBMD. ROC analysis for BAP and uNTX discriminated osteopenia/osteoporosis with AUC of 0.477 and 0.561, respectively. In the multivariable analysis, uNTX decreased with increasing trabecular fAGEs after adjusting for covariates (ß = 0.923;p = 0.031). CONCLUSION: This study demonstrated an inverse association of bone turnover markers and fAGEs. Both uNTX and BAP could not predict osteopenia/osteoporosis in the spine. uNTX reflects collagen characteristics and might have a complementary role to vBMD, as a non-invasive tool for bone quality assessment in spine surgery.

Intradermal Delivery of Calcium Hydroxylapatite With Fractionated Ablation.

OBJECTIVES: The absorption of biostimulatory particulate matter following its application to fractional skin defects remains poorly understood, and even less is known about its in vivo impact in terms of tissue integration. The objectives of this study are twofold: (1) to evaluate the potential of calcium hydroxylapatite (CaHA) to penetrate through skin treated with a fractional laser; and (2) to assess the effectiveness of clinical laser scanning microscopy technologies in monitoring the effects of such treatment over time. METHODS: One area on a volunteer's arm was treated with a fractional erbium laser (Sciton Inc., Palo Alto, CA), while a second area received the same laser treatment followed by CaHA topical application. We used reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) to noninvasively image beneath the surface of the treated skin to study and monitor the effects of these treatments within 1 h of treatment and at four additional time points over a 6-week period. RESULTS: One hour posttreatment, at different depths beneath the skin surface, MPM and RCM provided similar visualizations of laser-induced channels. In skin treated by both laser and CaHA, these two imaging methods provided complementary information. RCM captured the lateral and depth distribution of CaHA microspheres and were seen as bright spheres as they became incorporated into the healing tissue. MPM, meanwhile, visualized the CaHA microparticles as dark shadow spheres within the laser-induced channels and encroaching healing tissue. Furthermore, MPM provided critical information about collagen regeneration around the microspheres, with the collagen visually marked by its distinct second harmonic generation (SHG) signal. CONCLUSIONS: This observational pilot study demonstrates that CaHA, a collagen stimulator used as a dermal filler, can not only be inserted into the dermis after fractional laser treatment but remains in the healing skin for at least 6 weeks posttreatment. The noninvasive imaging techniques RCM and MPM successfully captured the presence of CaHA microspheres mid-dermis during the healing phase. They also demonstrated new collagen production around the microspheres, highlighting the effectiveness of these imaging approaches in monitoring such treatment over time.

Label-Free Monitoring of Endometrial Cancer Progression Using Multiphoton Microscopy.

Endometrial cancer is the most common gynecological cancer in the developed world. However, the accuracy of current diagnostic methods is still unsatisfactory and time-consuming. Here, we presented an alternate approach to monitoring the progression of endometrial cancer via multiphoton microscopy imaging and analysis of collagen, which is often overlooked in current endometrial cancer diagnosis protocols but can offer a crucial signature in cancer biology. Multiphoton microscopy (MPM) based on the second-harmonic generation and two-photon excited fluorescence was introduced to visualize the microenvironment of endometrium in normal, hyperplasia without atypia, atypical hyperplasia, and endometrial cancer specimens. Furthermore, automatic image analysis based on the MPM image processing algorithm was used to quantify the differences in the collagen morphological features among them. MPM enables the visualization of the morphological details and alterations of the glands in the development process of endometrial cancer, including irregular changes in the structure of the gland, increased ratio of the gland to the interstitium, and atypical changes in the glandular epithelial cells. Moreover, the destructed basement membrane caused by gland proliferation and fusion is clearly shown in SHG images, which is a key feature for identifying endometrial cancer progression. Quantitative analysis reveals that the formation of endometrial cancer is accompanied by an increase in collagen fiber length and width, a progressive linearization and loosening of interstitial collagen, and a more random arrangement of interstitial collagen. Observation and quantitative analysis of interstitial collagen provide invaluable information in monitoring the progression of endometrial cancer. Label-free multiphoton imaging reported here has the potential to become an in situ histological tool for effective and accurate early diagnosis and detection of malignant lesions in endometrial cancer.

Precise Serial Microregistration Enables Quantitative Microscopy Imaging Tracking of Human Skin Cells In Vivo.

We developed an automated microregistration method that enables repeated in vivo skin microscopy imaging of the same tissue microlocation and specific cells over a long period of days and weeks with unprecedented precision. Applying this method in conjunction with an in vivo multimodality multiphoton microscope, the behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, facilitating quantitative cellular dynamics analysis. We demonstrated the usefulness of this method in a skin biology study by successfully monitoring skin cellular responses for a period of two weeks following an acute exposure to ultraviolet light.

In vivo organoid growth monitoring by stimulated Raman histology.

Patient-derived tumor organoids have emerged as a crucial tool for assessing the efficacy of chemotherapy and conducting preclinical drug screenings. However, the conventional histological investigation of these organoids necessitates their devitalization through fixation and slicing, limiting their utility to a single-time analysis. Here, we use stimulated Raman histology (SRH) to demonstrate non-destructive, label-free virtual staining of 3D organoids, while preserving their viability and growth. This novel approach provides contrast similar to conventional staining methods, allowing for the continuous monitoring of organoids over time. Our results demonstrate that SRH transforms organoids from one-time use products into repeatable models, facilitating the efficient selection of effective drug combinations. This advancement holds promise for personalized cancer treatment, allowing for the dynamic assessment and optimization of chemotherapy treatments in patient-specific contexts.

Imaging of colorectal adenomas with pseudoinvasion and malignant polyps using two-photon excitation microscopy.

Introduction: Although the incidence and mortality rates of colorectal cancer exhibit significant variability, it remains one of the most prevalent cancers worldwide. Endeavors to prevent colorectal cancer development focus on detecting precursor lesions during colonoscopy. The diagnosis of endoscopically resected polyps relies on hematoxylin and eosin staining examination. For challenging cases like adenomatous polyps with epithelial misplacement, additional diagnostic methods could prove beneficial. Methods: This paper aims to underscore stromal changes observed in malignant polyps and polyps with pseudoinvasion, leveraging two-photon excitation microscopy (TPEM), a technique extensively employed in the medical field in recent years. Results and discussions: Both the subjective and quantitative analysis of TPEM images revealed distinct distributions and densities of collagen at the invasion front in malignant polyps compared to areas of pseudoinvasion. TPEM holds potential in discerning true invasion in malignant polyps from pseudoinvasion, offering enhanced visualization of local stromal changes.

A three-dimensional valve-on-chip microphysiological system implicates cell cycle progression, cholesterol metabolism and protein homeostasis in early calcific aortic valve disease progression.

BACKGROUND: Calcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. METHODS: In this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. RESULTS: Collagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. CONCLUSIONS: In this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. STATEMENT OF SIGNIFICANCE: Calcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment.

Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.

Label-free imaging diagnosis and collagen-optical evaluation of endometrioid adenocarcinoma with multiphoton microscopy.

The assessment of tumor grade and pathological stage plays a pivotal role in determining the treatment strategy and predicting the prognosis of endometrial cancer. In this study, we employed multiphoton microscopy (MPM) to establish distinctive optical pathological signatures specific to endometrioid adenocarcinoma (EAC), while also assessing the diagnostic sensitivity, specificity, and accuracy of MPM for this particular malignancy. The MPM technique exhibits robust capability in discriminating between benign hyperplasia and various grades of cancer tissue, with statistically significant differences observed in nucleocytoplasmic ratio and second harmonic generation/two-photon excited fluorescence intensity. Moreover, by utilizing semi-automated image analysis, we identified notable disparities in six collagen signatures between benign and malignant endometrial stroma. Our study demonstrates that MPM can differentiate between benign endometrial hyperplasia and EAC without labels, while also quantitatively assessing changes in the tumor microenvironment by analyzing collagen signatures in the endometrial stromal tissue.

Two-dimensional electro-optical multiphoton microscopy.

Significance: The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo. Aim: To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics. Approach: The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator. Results: Optimal KTN-EOD deflection of laser light with a central wavelength of 960 nm was obtained up to the highest average powers and pulse intensities tested (power: 350 mW; pulse duration: 118 fs). Up to 32 resolvable spots per line at a 560 kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a 13 µm by 13 µm field-of-view at over 10 kHz frame rate with ∼0.5 µm lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution. Conclusions: We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.

Large field-of-view metabolic profiling of murine brain tissue following morphine incubation using label-free multiphoton microscopy.

BACKGROUND: Although the effects on neural activation and glucose consumption caused by opiates such as morphine are known, the metabolic machinery underlying opioid use and misuse is not fully explored. Multiphoton microscopy (MPM) techniques have been developed for optical imaging at high spatial resolution. Despite the increased use of MPM for neural imaging, the use of intrinsic optical contrast has seen minimal use in neuroscience. NEW METHOD: We present a label-free, multimodal microscopy technique for metabolic profiling of murine brain tissue following incubation with morphine sulfate (MSO4). We evaluate two- and three-photon excited autofluorescence, and second and third harmonic generation to determine meaningful intrinsic contrast mechanisms in brain tissue using simultaneous label-free, autofluorescence multi-harmonic (SLAM) microscopy. RESULTS: Regional differences quantified in the cortex, caudate, and thalamus of the brain demonstrate region-specific changes to metabolic profiles measured from FAD intensity, along with brain-wide quantification. While the overall intensity of FAD signal significantly decreased after morphine incubation, this metabolic molecule accumulated near the nucleus accumbens. COMPARISON WITH EXISTING METHODS: Histopathology requires tissue fixation and staining to determine cell type and morphology, lacking information about cellular metabolism. Tools such as fMRI or PET imaging have been widely used, but lack cellular resolution. SLAM microscopy obviates the need for tissue preparation, permitting immediate use and imaging of tissue with subcellular resolution in its native environment. CONCLUSIONS: This study demonstrates the utility of SLAM microscopy for label-free investigations of neural metabolism, especially the intensity changes in FAD autofluorescence and structural morphology from third-harmonic generation.

Prognostic significance of collagen signatures in pancreatic ductal adenocarcinoma obtained from second-harmonic generation imaging.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) ranks among the deadliest types of cancer, and it will be meaningful to search for new biomarkers with prognostic value to help clinicians tailor therapeutic strategies. METHODS: Here we tried to use an advanced optical imaging technique, multiphoton microscopy (MPM) combining second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging, for the label-free detection of PDAC tissues from a cohort of 149 patients. An automated image processing method was used to extract collagen features from SHG images and the Kaplan-Meier survival analysis and Cox proportional hazards regression were used to assess the prognostic value of collagen signatures. RESULTS: SHG images clearly show the different characteristics of collagen fibers in tumor microenvironment. We gained eight collagen morphological features, and a Feature-score was derived for each patient by the combination of these features using ridge regression. Statistical analyses reveal that Feature-score is an independent factor, and can predict the overall survival of PDAC patients as well as provide well risk stratification. CONCLUSIONS: SHG imaging technique can potentially be a tool for the accurate diagnosis of PDAC, and this optical biomarker (Feature-score) may help clinicians make more approximate treatment decisions.

Quantification of age-related changes in the structure and mechanical function of skin with multiscale imaging.

The mechanical properties of skin change during aging but the relationships between structure and mechanical function remain poorly understood. Previous work has shown that young skin exhibits a substantial decrease in tissue volume, a large macro-scale Poisson's ratio, and an increase in micro-scale collagen fiber alignment during mechanical stretch. In this study, label-free multiphoton microscopy was used to quantify how the microstructure and fiber kinematics of aged mouse skin affect its mechanical function. In an unloaded state, aged skin was found to have less collagen alignment and more non-enzymatic collagen fiber crosslinks. Skin samples were then loaded in uniaxial tension and aged skin exhibited a lower mechanical stiffness compared to young skin. Aged tissue also demonstrated less volume reduction and a lower macro-scale Poisson's ratio at 10% uniaxial strain, but not at 20% strain. The magnitude of 3D fiber realignment in the direction of loading was not different between age groups, and the amount of realignment in young and aged skin was less than expected based on theoretical fiber kinematics affine to the local deformation. These findings provide key insights on how the collagen fiber microstructure changes with age, and how those changes affect the mechanical function of skin, findings which may help guide wound healing or anti-aging treatments.

Label-free, whole-brain in vivo mapping in an adult vertebrate with third harmonic generation microscopy.

Comprehensive understanding of interconnected networks within the brain requires access to high resolution information within large field of views and over time. Currently, methods that enable mapping structural changes of the entire brain in vivo are extremely limited. Third harmonic generation (THG) can resolve myelinated structures, blood vessels, and cell bodies throughout the brain without the need for any exogenous labeling. Together with deep penetration of long wavelengths, this enables in vivo brain-mapping of large fractions of the brain in small animals and over time. Here, we demonstrate that THG microscopy allows non-invasive label-free mapping of the entire brain of an adult vertebrate, Danionella dracula, which is a miniature species of cyprinid fish. We show this capability in multiple brain regions and in particular the identification of major commissural fiber bundles in the midbrain and the hindbrain. These features provide readily discernable landmarks for navigation and identification of regional-specific neuronal groups and even single neurons during in vivo experiments. We further show how this label-free technique can easily be coupled with fluorescence microscopy and used as a comparative tool for studies of other species with similar body features to Danionella, such as zebrafish (Danio rerio) and tetras (Trochilocharax ornatus). This new evidence, building on previous studies, demonstrates how small size and relative transparency, combined with the unique capabilities of THG microscopy, can enable label-free access to the entire adult vertebrate brain.

Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.