Light Sheet Fluorescence Microscopy Research Articles

Single-shot autofocusing in light sheet fluorescence microscopy with multiplexed structured illumination and deep learning

Light sheet fluorescence microscopy (LSFM) has been proposed as a promising tool for biological research due to its ability to observe the dynamics of living cells for hours and days. Nonetheless, this raises the strict requirement for the overlapping between the light sheet and the detection focal plane to get optimal image quality. Here, we describe a fast and accurate deep learning-based autofocusing method to overcome the challenge of unstable focusing in LSFM with a single shot. This method is compatible with any light sheet imaging setup with a spatial light modulator for light sheet generation. A predicted root-mean-square error of 0.0942 µm across a range of ±0.7 µm in a light sheet microscope using a 1.1 numerical aperture detection objective can be obtained. The neural network architecture we proposed shows advantages in small memory size, few training data set requirements, and good generalization to untrained sample types.

Beyond Early Development: Observing Zebrafish over 6 Weeks with Hybrid Optical and Optoacoustic Imaging

AbstractZebrafish are common model organisms in developmental biology, but have recently emerged as imaging targets of research in cancer, tissue regeneration, metabolic disorders, functional genomics, and phenotype‐based drug discovery. Conventionally, zebrafish are studied during the first few days of development using optical microscopy methods. However, optical methods are not suited for imaging at later stages, since the fish become opaque. To address needs to visualize beyond the first days of development, a novel multimodality system for observing zebrafish from larval stage to adulthood is developed. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy, fish (ex vivo) at stages of development up to 47 days at a similar object size‐to‐resolution ratio are imaged. Using multiple wavelength illumination over the visible and short‐wavelength infrared regions, it is demonstrated that the optoacoustic method can follow GFP‐based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover, the optoacoustic modality reveals zebrafish features based on optical contrast absent in SPIM, including contrast from endogenous blood, water, and lipids. It is discussed how the hybrid system method can enable the study of zebrafish in a wider range of applications and over time‐scales not possible currently when using optical microscopy.

4D Light-sheet imaging and interactive analysis of cardiac contractility in zebrafish larvae.

Despite ongoing efforts in cardiovascular research, the acquisition of high-resolution and high-speed images for the purpose of assessing cardiac contraction remains challenging. Light-sheet fluorescence microscopy (LSFM) offers superior spatiotemporal resolution and minimal photodamage, providing an indispensable opportunity for the in vivo study of cardiac micro-structure and contractile function in zebrafish larvae. To track the myocardial architecture and contractility, we have developed an imaging strategy ranging from LSFM system construction, retrospective synchronization, single cell tracking, to user-directed virtual reality (VR) analysis. Our system enables the four-dimensional (4D) investigation of individual cardiomyocytes across the entire atrium and ventricle during multiple cardiac cycles in a zebrafish larva at the cellular resolution. To enhance the throughput of our model reconstruction and assessment, we have developed a parallel computing-assisted algorithm for 4D synchronization, resulting in a nearly tenfold enhancement of reconstruction efficiency. The machine learning-based nuclei segmentation and VR-based interaction further allow us to quantify cellular dynamics in the myocardium from end-systole to end-diastole. Collectively, our strategy facilitates noninvasive cardiac imaging and user-directed data interpretation with improved efficiency and accuracy, holding great promise to characterize functional changes and regional mechanics at the single cell level during cardiac development and regeneration.

The influence of size and surface chemistry on the bioavailability, tissue distribution and toxicity of gold nanoparticles in zebrafish (Danio rerio)

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10–100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40–80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

3D open-top light-sheet microscopy and 3D microdissection of neuoadjuvant-treated primary prostate cancer reveals latent subclonal mutations.

e17109 Background: Intratumoral molecular heterogeneity is a well described phenomenon in primary prostate cancer. Increased genomic heterogeneity is associated with more aggressive clinical behavior, including recurrence and metastasis. However, current methods to characterize heterogeneity suffer from tradeoffs between generating high quality morphologic information, correlating morphology with genomic alterations, and workflow considerations. We describe a technology to image prostate samples in 3D, perform 3D microdissection of regions of interest (ROIs), and preserve nucleic acids for DNA sequencing. Methods: Samples (N = 18) were obtained from patients (N = 12) enrolled in an open-label, single-site, Phase II neoadjuvant trial in men with NCCN high to very-high risk PC. Patients received 12 weeks of apalutamide, abiraterone acetate plus prednisone, degarelix, and indomethacin followed by radical prostatectomy (RP). Fresh frozen prostate samples (~2x2x0.4cm) were obtained from RP specimens. Frozen samples were fixed in an ethanol-based fixative, stained with nuclear (TOPRO-3) and general protein (eosin) fluorescent dyes, cleared with ethyl cinnamate, and imaged using 3D open-top light-sheet (OTLS) fluorescence microscopy. Image-guided 3D microdissection was performed on ROIs with distinct morphologies. Microdissected ROIs measured 0.5x0.5x2mm. Microdissected samples were sent for DNA extraction and sequencing using the comprehensive clinically-validated UW-OncoPlex NGS assay. Results: The tissue from neoadjuvant-treated patients contained sparse carcinoma, with cancer volumes ranging from 0.01-0.3 cm2 and cellularity ranging from 2%-30%. Molecular alterations were detected in a sample with only 0.07 cm2 volume of carcinoma with 5% cellularity. The read depth of 3D microdissected samples was 3.9 times greater than the read depth of FFPE samples from the same patients. At least two morphologically distinct ROIs were 3D microdissected from 11 of 12 patients. Four of the 18 samples had molecular alterations that differed between ROIs, i.e. intra-sample molecular heterogeneity. For example, one sample with three microdissected ROIs had a PTEN alteration exclusive to a single ROI. Conclusions: The findings show the potential for 3D microdissection of morphologically distinct subclones in samples from neoadjuvant-treated patient, to enable sequencing of specific cellular populations. The sparsity of carcinoma necessitated a spatially targeted approach for extracting material for sequencing. The increased yield from avoiding FFPE processing and carcinoma enrichment from targeted 3D microdissection of carcinoma ROIs enabled the detection of spatially distinct molecular subclones. Future work will include the analysis of additional samples and implementation of AI-guided ROI detection to streamline workflows.

UNRAVELing the synergistic effects of psilocybin and environment on brain-wide immediate early gene expression in mice.

The effects of context on the subjective experience of serotonergic psychedelics have not been fully examined in human neuroimaging studies, partly due to limitations of the imaging environment. Here, we administered saline or psilocybin to mice in their home cage or an enriched environment, immunofluorescently-labeled brain-wide c-Fos, and imaged iDISCO+ cleared tissue with light sheet fluorescence microscopy (LSFM) to examine the impact of environmental context on psilocybin-elicited neural activity at cellular resolution. Voxel-wise analysis of c-Fos-immunofluorescence revealed clusters of neural activity associated with main effects of context and psilocybin-treatment, which were validated with c-Fos+ cell density measurements. Psilocybin increased c-Fos expression in subregions of the neocortex, caudoputamen, central amygdala, and parasubthalamic nucleus while it decreased c-Fos in the hypothalamus, cortical amygdala, striatum, and pallidum in a predominantly context-independent manner. To gauge feasibility of future mechanistic studies on ensembles activated by psilocybin, we confirmed activity- and Cre-dependent genetic labeling in a subset of these neurons using TRAP2+/-;Ai14+ mice. Network analyses treating each psilocybin-sensitive cluster as a node indicated that psilocybin disrupted co-activity between highly correlated regions, reduced brain modularity, and dramatically attenuated intermodular co-activity. Overall, our results indicate that main effects of context and psilocybin were robust, widespread, and reorganized network architecture, whereas context×psilocybin interactions were surprisingly sparse.

Deep learning-based adaptive optics for light sheet fluorescence microscopy.

Light sheet fluorescence microscopy (LSFM) is a high-speed imaging technique that is often used to image intact tissue-cleared specimens with cellular or subcellular resolution. Like other optical imaging systems, LSFM suffers from sample-induced optical aberrations that decrement imaging quality. Optical aberrations become more severe when imaging a few millimeters deep into tissue-cleared specimens, complicating subsequent analyses. Adaptive optics are commonly used to correct sample-induced aberrations using a deformable mirror. However, routinely used sensorless adaptive optics techniques are slow, as they require multiple images of the same region of interest to iteratively estimate the aberrations. In addition to the fading of fluorescent signal, this is a major limitation as thousands of images are required to image a single intact organ even without adaptive optics. Thus, a fast and accurate aberration estimation method is needed. Here, we used deep-learning techniques to estimate sample-induced aberrations from only two images of the same region of interest in cleared tissues. We show that the application of correction using a deformable mirror greatly improves image quality. We also introduce a sampling technique that requires a minimum number of images to train the network. Two conceptually different network architectures are compared; one that shares convolutional features and another that estimates each aberration independently. Overall, we have presented an efficient way to correct aberrations in LSFM and to improve image quality.

Novel discovery of a lymphatic bridge connecting Schlemm's canal to limbal and conjunctival lymphatic pathway

PurposeSchlemm's canal (SC) is a critical structure regulating aqueous humor (AH) drainage and intraocular pressure (IOP). It is known that in the conventional outflow pathway, AH flows from SC to episcleral veins. We recently reported a high-resolution three-dimensional (3D) imaging technology for intact eyeballs, SC and ocular surface. Using this advanced technology, we herein report the discovery of a new structure, termed lymphatic bridge, that directly connects SC to the limbal and conjunctival lymphatic pathway. Further investigation on this novel outflow pathway may provide new mechanisms and therapeutic approaches for glaucoma. MethodsAs reported previously, intact eyeballs were harvested from Prox-1-GFP (green fluorescent protein) mice and processed by a tissue clearing technique with CLARITY. Samples were immunolabeled with specific antibodies for CD31 (pan-endothelial marker) and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) and imaged by light-sheet fluorescent microscopy. The limbal areas were examined to locate connecting channels between SC and limbal and conjunctival lymphatic vessels. Moreover, in vivo anterior chamber dye injection was performed with Texas Red dextran for functional analysis on AH outflow. ResultsA novel lymphatic bridge structure that expressed both Prox-1 and LYVE-1 was discovered between the SC and limbal lymphatic vessels connected with conjunctival lymphatic pathway. Results from the anterior chamber dye injection also confirmed AH drainage into the conjunctival lymphatic outflow pathway. ConclusionsThis study provides the first evidence on the direct connection between SC and conjunctival lymphatic pathway. This new pathway is distinctive from the traditional episcleral vein pathway and merits further investigation.

60 Multimodal Imaging and Artificial Intelligence Unveil Hot-Spot Deposition, Bronchial/Alveolar Dose and Cellular Fate of Biopersistent Nanoparticles in the Lung

Abstract Pulmonary delivery is not only of increasing importance to exploit local therapy for lung diseases like COVID-19, but also plays an essential role in interpreting health impact of inhaled hazardous (nano-)materials. Deciphering of biodistribution, biokinetics, and cell-mediated immunity of pulmonary delivered nanoparticles (NPs) remains insufficient. We applied state-of-the-art multimodal imaging and deep learning pipelines to elucidate the intricate deposition profiles and the fate of biopersistent NPs in the lung within cellular resolution. Spatially resolved delivery characteristics like inter- and intra-acinar distribution pattern and regional NP dosage were revealed by tissue-clearing light-sheet fluorescence microscopy for six common routes of pulmonary delivery, i.e., intranasal & oropharyngeal aspiration, intubated instillation, microsprayer application, ventilator-assisted & nose-only aerosol inhalation. Artificial intelligence-driven entire airway 3D segmentation (convolutional neural network) enabled accurate dosimetry of bronchial versus acinar/alveolar NP dose (B/A: 0.04 to 0.18 depending on delivery route) rather than the conventionally simplified 2D central-to-peripheral ratio (C/P: 1.32 to 3.79). Moreover, longitudinal 3D imaging revealed similar intra-acinar NP kinetics independent of delivery route. Inhaled NP relocation from the alveolar-epithelium to interstitium was traditionally claimed as the consequence of passive diffusion/transportation of NPs. This study, however, revealed that acinar NP transport can be attributed to the phagocytosis and migration of tissue-resident macrophages (TRMs) as evidenced by complementary analytical approaches (intravital microscopy, ex vivo 4D live-tissue microscopy, in situ immunofluorescence, ex vivo NP-lung interaction model, flow cytometry). Overall, this study elucidates holistic NP-lung delivery and biokinetics features and TRM immunity on the fate of biopersistent NPs.

Acoustic prison for single live cell 3D multi-imaging enabled by light-sheet microscopy

Having a system capable of obtaining high resolution, quantitative, subcellular information of individual live cells through imaging, while minimizing cell damage, is of great importance in many fields of research. In recent years, optofluidic light-sheet fluorescent microscopy (LSFM) has emerged as a powerful tool providing a low photo-toxic imaging method utilising the fluidic environment offered by microfluidics. Here, the benefits of LSFM were integrated with an acoustic single cell prison for precise single cell handling and 3D multi-imaging in a semi-automated manner. The compact, monolithic, acousto-optofluidic platform employed standing surface acoustic waves (SSAWs) to sequentially trap an individual cell on either side of an imaging region, which gathered planar, cross-sectional images of the cell. A cytoplasmic stain was utilised to not only visualize the cell trajectory throughout the imaging process, but also to verify the cell viability post-acoustic exposure. A mitochondrial stain was also used to better demonstrate the resolution capabilities of the device. Through post-image processing, 3D volumetric images of the cell were reconstructed and the results between the first and second rounds of imaging were directly comparable. Furthermore, the acoustic prison advantageously positions the cells in the upper region of the channel, enabling the ability to accurately compare temporal changes in cell morphology; a capability that can lead to advancements in therapeutics and drug delivery to access responses of cells to stimuli over time.

OptoRheo: Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named ‘OptoRheo’ that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Multi-Resolution 3D Optical Mapping of Immune Cell Infiltrates in Mouse Asthmatic Lung.

Asthma is a chronic inflammatory airway disease driven by various infiltrating immune cell types into the lung. Optical microscopy has been used to study immune infiltrates in the asthmatic lungs. Confocal laser scanning microscopy (CLSM) identifies the phenotypes and locations of individual immune cells in lung tissue sections by employing high-magnification objectives and multiplex immunofluorescence staining. In contrast, light-sheet fluorescence microscopy (LSFM) can visualize the macroscopic and mesoscopic architecture of whole-mount lung tissues in three dimensions (3D) by adopting an optical tissue clearing method. Despite each microscopy method producing image data with unique resolution from a tissue sample, CLSM and LSFM have not been applied together due to the difference in the tissue preparation procedures. Here we introduce a new approach combining LSFM and CLSM into a sequential imaging pipeline. We built a new optical tissue clearing workflow in which the immersion clearing agent can be switched from an organic solvent to an aqueous sugar solution for sequential 3D LSFM and CLSM of mouse lungs. This sequential combination microscopy offered quantitative 3D spatial analyses of the distribution of immune infiltrates in the same mouse asthmatic lung tissue at the organ, tissue, and cell level. These results show that our method facilitates multi-resolution 3D fluorescence microscopy as a new imaging approach providing comprehensive spatial information for a better understanding of inflammatory lung diseases. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Perineurial Window is Critical for Experimental Reverse End-to-Side Nerve Transfer.

The depth of connective tissue window in the side of a recipient nerve in reverse end-to-side transfers (RETS) remains controversial. To test whether the depth of connective tissue disruption influences the efficiency of donor axonal regeneration in the context of RETS. Sprague-Dawley rats (n = 24) were assigned to 1 of the 3 groups for obturator nerve to motor femoral nerve RETS: group 1, without epineurium opening; group 2, with epineurium only opening; and group 3, with epineurium and perineurium opening. Triple retrograde labeling was used to assess the number of motor neurons that had regenerated into the recipient motor femoral branch. Thy1-GFP rats (n = 8) were also used to visualize the regeneration pathways in the nerve transfer networks at 2- and 8-week time point using light sheet fluorescence microscopy. The number of retrogradely labeled motor neurons that had regenerated distally toward the target muscle was significantly higher in group 3 than that in groups 1 and 2. Immunohistochemistry validated the degree of connective tissue disruption among the 3 groups, and optical tissue clearing methods demonstrated donor axons traveling outside the fascicles in groups 1 and 2 but mostly within the fascicles in group 3. Creating a perineurial window in the side of recipient nerves provides the best chances of robust donor axonal regeneration across the RETS repair site. This finding aids nerve surgeons by confirming that a deep window should be undertaken when doing a RETS procedure.

COMBINe enables automated detection and classification of neurons and astrocytes in tissue-cleared mouse brains

Tissue clearing renders entire organs transparent to accelerate whole-tissue imaging; for example, with light-sheet fluorescence microscopy. Yet, challenges remain in analyzing the large resulting 3D datasets that consist of terabytes of images and information on millions of labeled cells. Previous work has established pipelines for automated analysis of tissue-cleared mouse brains, but the focus there was on single-color channels and/or detection of nuclear localized signals in relatively low-resolution images. Here, we present an automated workflow (COMBINe, Cell detectiOn in Mouse BraIN) to map sparsely labeled neurons and astrocytes in genetically distinct mouse forebrains using mosaic analysis with double markers (MADM). COMBINe blends modules from multiple pipelines with RetinaNet at its core. We quantitatively analyzed the regional and subregional effects of MADM-based deletion of the epidermal growth factor receptor (EGFR) on neuronal and astrocyte populations in the mouse forebrain.

Active mesh and neural network pipeline for cell aggregate segmentation

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.

Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth.

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Advanced microscopy resolves dynamic localization patterns of stress-induced mitogen-activated protein kinase (SIMK) during alfalfa root hair interactions with Ensifer meliloti.

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

Image-based modeling of vascular organization to evaluate anti-angiogenic therapy

In tumor therapy anti-angiogenic approaches have the potential to increase the efficacy of a wide variety of subsequently or co-administered agents, possibly by improving or normalizing the defective tumor vasculature. Successful implementation of the concept of vascular normalization under anti-angiogenic therapy, however, mandates a detailed understanding of key characteristics and a respective scoring metric that defines an improved vasculature and thus a successful attempt. Here, we show that beyond commonly used parameters such as vessel patency and maturation, anti-angiogenic approaches largely benefit if the complex vascular network with its vessel interconnections is both qualitatively and quantitatively assessed. To gain such deeper insight the organization of vascular networks, we introduce a multi-parametric evaluation of high-resolution angiographic images based on light-sheet fluorescence microscopy images of tumors. We first could pinpoint key correlations between vessel length, straightness and diameter to describe the regular, functional and organized structure observed under physiological conditions. We found that vascular networks from experimental tumors diverted from those in healthy organs, demonstrating the dysfunctionality of the tumor vasculature not only on the level of the individual vessel but also in terms of inadequate organization into larger structures. These parameters proofed effective in scoring the degree of disorganization in different tumor entities, and more importantly in grading a potential reversal under treatment with therapeutic agents. The presented vascular network analysis will support vascular normalization assessment and future optimization of anti-angiogenic therapy.

Combined Chemoradionuclide Therapy Using Poly(ε‐caprolactone‐b‐ethylene oxide) Micelles as the Delivery Vehicle

AbstractCombination of therapies is a common strategy in cancer treatment. Such combined therapies only have merit provided that there is superior therapeutic outcome with fewer side effects, compared to single therapies. Here, this work explores the possibility to combine chemotherapy with radionuclide therapy using polymeric micelles as a delivery vehicle. For this purpose, this work prepares poly(ε‐caprolactone‐b‐ethylene oxide) (PCL‐PEO) micelles and load them simultaneously with paclitaxel (PTX) and 177Lu(III). This work chooses a 3D tumor spheroid composed of glioblastoma cells (U87) to evaluate the combined treatment. The diffusion of the micelles in the spheroid is investigated by confocal laser scanning microscopy (CLSM) and light‐sheet fluorescence microscopy (LSFM). The results show that the micelles are able to penetrate deep into the spheroid within 24 h of incubation and mainly accumulated around or in the lysosomes once in the cell. Subsequently, this work evaluates the cell killing efficiency of the single treatments (PTX or 177Lu(III)) versus combined treatment (PTX + 177Lu(III)) by measuring the growth of the spheroids as well as by performing a cell‐viability assay. The results indicate that the combined therapy achieves a superior therapeutic outcome with better cell growth inhibition and cell killing efficiency compared to the single treatments.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.