Confocal Imaging System Research Articles

In vivo observation of multi-phase spatiotemporal cellular dynamics of transplanted HSPCs during early engraftment.

Hematopoietic stem cell transplantation (HSCT) is commonly used to treat patients with various blood disorders, genetic and immunological diseases, and solid tumors. Several systemic complications following HSCT are critical limiting factors for achieving a successful outcome. These systemic complications are mainly due to the lack of initial engraftment after transplantation. However, the detailed underlying cellular dynamics of early engraftment have not been fully characterized yet. We performed in vivo longitudinal visualization of early engraftment characteristics of transplanted hematopoietic stem and progenitor cells (HSPCs) in the mouse calvarial bone marrow (BM). To achieve this, we utilized an in vivo laser‐scanning confocal microscopy imaging system with a cranial BM imaging window and stereotaxic device. We observed two distinct cellular behaviors of HSPCs in vivo, cluster formation and cluster dissociation, early after transplantation. Furthermore, we successfully identified three cellular phases of engraftment with distinct cellular distances which are coordinated with cell proliferation and cell migration dynamics during initial engraftment.

Noninvasive Measurement of Oxygen Saturation in Human Retinal Blood Vessels and Tissues With Multispectral Confocal Imaging.

Proof of concept for the first system of noninvasive human retinal vessel and tissue oxygenation measurement in axial and radial directions. A confocal imaging system capable of calculating and mapping relative retinal blood oxygenation in radial and axial directions from three eyes of two healthy subjects was built. The relationship between oxygenation and retinal depth in vivo was analyzed to illustrate application of this novel system. The system shows capacity for measuring oxygenation along retinal depth for the first time. (1) Arteriovenous oxygenation difference decreases with blood vessel diameter. (2) Artery-tissue oxygenation difference is greater than vein-tissue oxygenation difference in the same region. (3) Intravascular-extravascular oxygenation difference decreases with blood vessel diameter. (4) Oxygenation data reported with a 95% CI are as follows: A1 91.5% ± 18.2%, V1 32.8% ± 18.6%, A2 97.3% ± 17.8%, V2 64.4% ± 11.2%, A3 73.2% ± 19.1%, V3 52.9% ± 15.3%, and Tissue 56.6% ± 00.4%. This article demonstrates proof of concept for retinal oxygenation calculation in radial and axial dimensions for the first time. Initial results provide biological validity to this method. Future aims include further characterization of this system's results in healthy subjects and subsequent comparison of oxygenation between diseased and healthy retinae. [Ophthalmic Surg Lasers Imaging Retina. 2022;53:275-283.].

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Handheld laser scanning microscope catheter for real-time and in vivo confocal microscopy using a high definition high frame rate Lissajous MEMS mirror.

A handheld confocal microscope using a rapid MEMS scanning mirror facilitates real-time optical biopsy for simple cancer diagnosis. Here we report a handheld confocal microscope catheter using high definition and high frame rate (HDHF) Lissajous scanning MEMS mirror. The broad resonant frequency region of the fast axis on the MEMS mirror with a low Q-factor facilitates the flexible selection of scanning frequencies. HDHF Lissajous scanning was achieved by selecting the scanning frequencies with high greatest common divisor (GCD) and high total lobe number. The MEMS mirror was fully packaged into a handheld configuration, which was coupled to a home-built confocal imaging system. The confocal microscope catheter allows fluorescence imaging of in vivo and ex vivo mouse tissues with 30 Hz frame rate and 95.4% fill factor at 256 × 256 pixels image, where the lateral resolution is 4.35 μm and the field-of-view (FOV) is 330 μm × 330 μm. This compact confocal microscope can provide diverse handheld microscopic applications for real-time, on-demand, and in vivo optical biopsy.

Spatial coherence of electrically pumped random terahertz lasers

Light sources with high radiance and tailored coherence properties are highly desirable for imaging applications in the mid-infrared and terahertz (THz) spectral regions, which host a large variety of molecular absorptions and distinctive fingerprints to be exploited for sensing and tomography. Here, we characterize the spatial coherence of random multimode THz quantum cascade lasers (QCLs) emitting > mW optical power per mode and showing low divergence (10°–30°), performing a modified Young’s double-slit experiment. Partial spatial coherence values ranging between 0.16 and 0.34 are retrieved, depending on the specific degree of disorder. These values are significantly lower than those (0.82) of conventional Fabry–Perot THz QCLs exploiting an identical active region quantum design. We then incorporate the devised low spatial coherence random lasers into a confocal imaging system with micrometer spatial resolution and demonstrate notable imaging performances, at THz frequencies, against spatial cross talk and speckles.

Computational adaptive optics for high-resolution non-line-of-sight imaging.

Non-line-of-sight (NLOS) imaging has aroused great interest during the past few years, by providing a unique solution for the observation of hidden objects behind obstructions or scattering media. As such, NLOS imaging may facilitate broad applications in autonomous driving, remote sensing, and medical diagnosis. However, existing NLOS frameworks suffer from severe degradation of resolution and signal-to-noise ratio (SNR) due to aberrations induced by scattering media and system misalignment, restricting its practical applications. This paper proposes a computational adaptive optics (CAO) method for NLOS imaging to correct optical aberrations in post-processing without the requirement of any hardware modifications. We demonstrate the effectiveness of CAO with a confocal NLOS imaging system in Terahertz (THz) band by imaging different samples behind occlusions for both low- and high-order aberrations. With appropriate metrics used for iterative CAO in post-processing, both the resolution and SNR can be increased by several times without reducing the data acquisition speed.

Super-resolution imaging based on radially polarized beam induced superoscillation using an all-dielectric metasurface.

Superoscillation is a kind of phenomenon which can generate oscillation faster than the fastest component of a band-limited function. For optics, superoscillation is generated by coherence of low spatial frequency waves. It can bring a localized region named "hot spot", which has a smaller size than the diffraction-limit, and this character has potential applicaions in super-resolution imaging. Using a high-order radially polarized Laguerre-Gaussian beam tightly focused by high-NA objective lens, we can easily obtain and control the superoscillation hot spot. Using a metasurface, which has compact volume and sub-wavelength pixel size, we can generate the high-order radially polarized Laguerre-Gaussian beam more simply than conventional methods like using a liquid crystal mode converter. We first analyze the properties of unit cells of the metasurface and simulate the performance of the metasurface. Then we analyze the property of the tightly focused high-order radially polarized Laguerre-Gaussian beam and design a super-resolution imaging system using our designed metasurface. Therefore, the 2-fold lateral resolution enhancement is realized in our approach. This method can be used to improve lateral resolution in conventional confocal imaging systems.

TMOD-13. IDENTIFYING DRIVERS IN THE CONVERGING SYNTENIC REGIONS OF SPONTANEOUS CANINE AND PEDIATRIC HIGH-GRADE GLIOMA USING IMAGING BASED CRISPR-CAS9 ARRAY SCREEN

Abstract Gliomas occur in companion dogs at rates comparable to humans, with short-snouted breeds such as boxers being more susceptible than others. The natural progression of cancer in the immuno-competent host allows companion dogs diagnosed with sporadic glioma as an optimal model for preclinical testing of therapeutic approaches with human relevance, including immunotherapies. We have recently performed comprehensive genomic and epigenetic characterization of glioma in dogs to their human counterparts and found strong convergent evolution – shared somatic mutations and aneuploidies - among syntenic regions, including those of known pediatric glioma drivers, e.g., PDGFRA, MYC, PIK3CA. Here, using arrayed CRISPR-Cas9 imaging based phenotypic screen, we will probe potential oncogenic drivers and tumor suppressor genes within syntenic aneuploidies and thus outline functional versus non-functional heterogeneity of cancer aneuploidy. Specifically, we are conducting arrayed knockout screen (one gene per well) of 400+ genes within syntenic aneuploidies across canine (n=2) and pediatric (n=2) high-grade glioma cell lines. We will first capture images by high-speed confocal imaging system at three time points post-transduction of single guide RNAs (2 per gene) targeting each of 400+ genes in their separate wells. Then, using high-throughput image analysis and semi-supervised machine learning methods, we will measure well-based phenotypic features (viability, growth, and morphology) from these images. Genes will be ranked per cross-validated predicted probability in yielding either proliferating or slow-growing cell type based on learned phenotypic features using image data of knockout cells from and across wells. The top ranked genes will then be linked to oncogenes and tumor suppressors based on pathway and ontology analysis. We expect that we will see convergence of the most impactful molecular abnormalities (based on their knockout phenotypes) on mechanisms or candidate signaling pathways for the development of new drugs and repurposing of existing drugs for kids and dogs with high-grade glioma.

Fluorescence-Based Real-Time Analysis of Osteoclast Development.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

Abstract 5: Experimentally-driven mathematical model of tumor angiogenesis mediated by multiple angiogenic factors

Abstract Tumor angiogenesis is regulated by multiple pro- and anti-angiogenic factors. Anti-angiogenic drugs target the interconnected network of these angiogenic factors to inhibit neovascularization and tumor growth. However, current anti-angiogenic therapeutics targeting a single angiogenic factor show limited clinical success, prompting the development of combination anti-angiogenic therapy targeting multiple angiogenic factors simultaneously. In cases where anti-angiogenic therapy may be effective, tumors display a wide range of responses, which is not fully understood. In this work, we use an integrative in vitro imaging and mathematical modeling approach to investigate how tumor angiogenesis is systematically regulated by multiple angiogenic factors and to study the effects of anti-angiogenic therapy. We particularly focus on two pro-angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2), and two anti-angiogenic factors, thrombospondin-1 (TSP1) and platelet factor 4 (PF4). In our in vitro experimental setting human endothelial cells are cultured in a microfluidic organ-on-chip chip platform exposed to angiogenic factors or in the presence of cancer cells to mimic the tumor microenvironment. The cellular responses induced by various concentrations of angiogenic factors are measured through a high content screening (HCS) confocal imaging system to quantify the cell counts, morphological features and vessel characteristics. These data are used to construct a novel mathematical model to capture how the angiogenic factors mediate cross-talk between tumor cells and endothelial cells. By fitting the model to the experimental measurements, we are able to predict the temporal dynamics of cell numbers and concentrations of the angiogenic factors. Using the model, we generate insights into the effects of angiogenic factors and various anti-angiogenic treatments on tumor cells and endothelial cells. Excitingly, the model can also be applied to investigate how the effect of anti-angiogenic therapy is influenced by inter-tumor heterogeneity. For example, we identified that tumors with lower tumor cell growth rate and higher carrying capacity have a stronger response to anti-VEGF treatment, which indicates that variation in tumor cell growth rates can be a main reason for the observed heterogenous response to anti-VEGF therapy. In addition, we investigate a novel hypothesis regarding synergy between anti-angiogenic and chemotherapeutic agents with the model. Our simulated results suggest a new mechanism in which the chemotherapeutic agent enhances anti-angiogenic therapy simply through reducing the tumor cell growth rate. Overall, this work generates novel insights into the function of multiple angiogenic factors in tumor angiogenesis, providing a tool that can be further used to test and optimize anticancer therapy. Citation Format: Ding Li, Danielle D. Hixon, Shannon M. Mumenthaler, Stacey D. Finley. Experimentally-driven mathematical model of tumor angiogenesis mediated by multiple angiogenic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 5.

Calcium Imaging Analysis of Cellular Responses to Hypercapnia and Hypoxia in the NTS of Newborn Rat Brainstem Preparation.

It is supposed that the nucleus of the solitary tract (NTS) in the dorsal medulla includes gas sensor cells responsive to hypercapnia or hypoxia in the central nervous system. In the present study, we analyzed cellular responses to hypercapnia and hypoxia in the NTS region of newborn rat in vitro preparation. The brainstem and spinal cord were isolated from newborn rat (P0-P4) and were transversely cut at the level of the rostral area postrema. To detect cellular responses, calcium indicator Oregon Green was pressure-injected into the NTS just beneath the cut surface of either the caudal or rostral block of the medulla, and the preparation was superfused with artificial cerebrospinal fluid (25–26°C). We examined cellular responses initially to hypercapnic stimulation (to 8% CO2 from 2% CO2) and then to hypoxic stimulation (to 0% O2 from 95% O2 at 5% CO2). We tested these responses in standard solution and in two different synapse blockade solutions: (1) cocktail blockers solution including bicuculline, strychnine, NBQX and MK-801 or (2) TTX solution. At the end of the experiments, the superfusate potassium concentration was lowered to 0.2 from 3 mM to classify recorded cells into neurons and astrocytes. Excitation of cells was detected as changes of fluorescence intensity with a confocal calcium imaging system. In the synaptic blockade solutions (cocktail or TTX solution), 7.6 and 8% of the NTS cells responded to hypercapnic and hypoxic stimulation, respectively, and approximately 2% of them responded to both stimulations. Some of these cells responded to low K+, and they were classified into astrocytes comprising 43% hypercapnia-sensitive cells, 56% hypoxia-sensitive cells and 54% of both stimulation-sensitive cells. Of note, 49% of the putative astrocytes identified by low K+ stimulation were sensitive to hypercapnia, hypoxia or both. In the presence of a glia preferential blocker, 5 mM fluoroacetate (plus 0.5 μM TTX), the percentage of hypoxia-sensitive cells was significantly reduced compared to those of all other conditions. This is the first study to reveal that the NTS includes hypercapnia and hypoxia dual-sensitive cells. These results suggest that astrocytes in the NTS region could act as a central gas sensor.

Regulation of smooth muscle contractility by the epithelium in rat tracheas: role of prostaglandin E2 induced by the neurotransmitter acetylcholine.

BackgroundPrevious studies have suggested the involvement of epithelium in modulating the contractility of neighboring smooth muscle cells. However, the mechanism underlying epithelium-derived relaxation in airways remains largely unclear. This study aimed to investigate the mechanism underlying epithelium-dependent smooth muscle relaxation mediated by neurotransmitters.MethodsThe contractile tension of Sprague-Dawley (SD) rat tracheal rings were measured using a mechanical recording system. Intracellular Ca2+ level was measured using a Ca2+ fluorescent probe Fluo-3 AM, and the fluorescence signal was recorded by a laser scanning confocal imaging system. The prostaglandin E2 (PGE2) content was measured using an enzyme-linked immunosorbent assay kit.ResultsWe observed that the neurotransmitter acetylcholine (ACh) restrained the electric field stimulation (EFS)-induced contraction in the intact but not epithelium-denuded rat tracheal rings. After inhibiting the muscarinic ACh receptor (mAChR) or cyclooxygenase (COX), a critical enzyme in prostaglandin synthesis, the relaxant effect of ACh was attenuated. Exogenous PGE2 showed a similar inhibitory effect on the EFS-evoked contraction of tracheal rings. Moreover, ACh triggered phospholipase C (PLC)-coupled Ca2+ release from intracellular Ca2+ stores and stimulated COX-dependent PGE2 production in primary cultured rat tracheal epithelial cells.ConclusionsCollectively, this study demonstrated that ACh induced rat tracheal smooth muscle relaxation by promoting PGE2 release from tracheal epithelium, which might provide valuable insights into the cross-talk among neurons, epithelial cells and neighboring smooth muscle cells in airways.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

BackgroundThe inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.ResultsConventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.ConclusionWe present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

Fast confocal Raman imaging via context-aware compressive sensing

Raman hyperspectral imaging is a powerful method to obtain detailed chemical information about a wide variety of organic and inorganic samples noninvasively and without labels. However, due to the weak, nonresonant nature of spontaneous Raman scattering, acquiring a Raman imaging dataset is time-consuming and inefficient. In this paper we utilize a compressive imaging strategy coupled with a context-aware image prior to improve Raman imaging speed by 5- to 10-fold compared to classic point-scanning Raman imaging, while maintaining the traditional benefits of point scanning imaging, such as isotropic resolution and confocality. With faster data acquisition, large datasets can be acquired in reasonable timescales, leading to more reliable downstream analysis. On standard samples, context-aware Raman compressive imaging (CARCI) was able to reduce the number of measurements by ∼85% while maintaining high image quality (SSIM >0.85). Using CARCI, we obtained a large dataset of chemical images of fission yeast cells, showing that by collecting 5-fold more cells in a given experiment time, we were able to get more accurate chemical images, identification of rare cells, and improved biochemical modeling. For example, applying VCA to nearly 100 cells' data together, cellular organelles were resolved that were not faithfully reconstructed by a single cell's dataset.

“Two-Cell Assemblage” Assay: A Simple in vitro Method for Screening Hair Growth-Promoting Compounds

Alopecia arises due to inadequate hair follicle (HF) stem cell activation or proliferation, resulting in prolongation of the telogen phase of the hair cycle. Increasing therapeutic and cosmetic demand for alleviating alopecia has driven research toward the discovery or synthesis of novel compounds that can promote hair growth by inducing HF stem cell activation or proliferation and initiating the anagen phase. Although several methods for evaluating the hair growth-promoting effects of candidate compounds are being used, most of these methods are difficult to use for large scale simultaneous screening of various compounds. Herein, we introduce a simple and reliable in vitro assay for the simultaneous screening of the hair growth-promoting effects of candidate compounds on a large scale. In this study, we first established a 3D co-culture system of human dermal papilla (hDP) cells and human outer root sheath (hORS) cells in an ultra-low attachment 96-well plate, where the two cell types constituted a polar elongated structure, named “two-cell assemblage (TCA).” We observed that the long axis length of the TCA gradually increased for 5 days, maintaining biological functional integrity as reflected by the increased expression levels of hair growth-associated genes after treatment with hair growth-promoting molecules. Interestingly, the elongation of the TCA was more prominent following treatment with the hair growth-promoting molecules (which occurred in a dose-dependent manner), compared to the control group (p < 0.05). Accordingly, we set the long axis length of the TCA as an endpoint of this assay, using a micro confocal high-content imaging system to measure the length, which can provide reproducible and reliable results in an adequate timescale. The advantages of this assay are: (i) it is physiologically and practically advantageous as it uses 3D cultured two-type human cells which are easily available; (ii) it is simple as it uses length as the only endpoint; and (iii) it is a high throughput system, which screens various compounds simultaneously. In conclusion, the “TCA” assay could serve as an easy and reliable method to validate the hair growth-promoting effect of a large volume of library molecules.

Abstract 16955: Contrasting Effects of Smooth Muscle TRPV4 Channels on Vascular Function

Introduction: Transient receptor potential vanilloid 4 (TRPV4) channels are a major Ca 2+ entry pathway in vascular endothelial cells. TRPV4 channels are also expressed in vascular smooth muscle cells (SMC). However, the effect of SMC TRPV4 (TRPV4 SMC ) channels on vascular resistance and blood pressure remains controversial. Hypothesis: We hypothesized that TRPV4 SMC channels are essential regulators of vascular function and blood pressure. Methods: Inducible, SMC-specific TRPV4 knockout (TRPV4 SMC -/- ) mice were used. Blood pressure was monitored using radiotelemetry catheters. SMC Ca 2+ signals were recorded with a spinning disk confocal imaging system. Pressure myography studies assessed vascular reactivity in MAs. Results: Resting diastolic blood pressure and mean arterial pressure were lower in TRPV4 SMC -/- mice when compared to control mice. Selective TRPV4 channel agonist, GSK1016790A (GSK101), induced Ca 2+ influx signals in SMCs that were inhibited by TRPV4 channel inhibitor GSK2193874 and were absent in MAs from TRPV4 SMC -/- mice. In pressure myography studies, GSK101 constricted the MAs in control mice, but not in TRPV4 SMC -/- mice. Moreover, MAs from TRPV4 SMC -/- showed lower vasoconstriction in response to phenylephrine, a α1 adrenergic receptor agonist. These results indicated that Ca 2+ influx through TRPV4 SMC channels contributes to vasoconstriction and increased vascular resistance. However, studies of pressure-induced constriction or myogenic tone indicated that MAs from TRPV4 SMC -/- mice exhibit a higher myogenic constriction at pressures ≥ 60 mm Hg. Moreover, membrane depolarization-induced vasoconstriction (KCl, 33 and 60 mM) was also increased in TRPV4 SMC -/- mice when compared to the control mice, indicating that TRPV4 SMC channels oppose pressure- or membrane depolarization-induced vasoconstriction. Conclusions: TRPV4 SMC channels are essential regulators of resting blood pressure. While TRPV4 SMC channels contribute to α1 adrenergic receptor-induced vasoconstriction, they negatively regulate pressure-induced vasoconstriction. The divergent effects of TRPV4 SMC channels on vascular function may be determined by the selective stimulus-TRPV4 SMC channel coupling and downstream signaling mechanisms.

An in vitro study on the antitumor effect of sonodynamic therapy using sinoporphyrin sodium on human glioblastoma cells

Sonodynamic therapy (SDT) is a promising modality for cancer treatment. Sinoporphyrin sodium (DVDMS), purified from Photofrin II, shows great potential in SDT evidenced by growing studies. The purpose of the current study was to investigate the antitumor effect of SDT combined with DVDMS on human glioblastoma (U87 MG) cell line in vitro. The cellular uptake of DVDMS was investigated by confocal microscopy and IVIS spectrum imaging system. In addition, DVDMS toxicity and anti-tumor effect of SDT were assessed by flow cytometry. The generation of intracellular reactive oxygen species (ROS) was determined using DCFH-DA staining. Simultaneously, fluorescence microscopy was performed to access the destabilization of mitochondrial membrane potential (MMP). The results showed that DVDMS could easily enter the cells and accumulated in the cytoplasm, especially the mitochondria. And the intracellular DVDMS increased with incubation time or concentrations. The results also showed remarkable cytotoxicity of DVDMS-mediated SDT (center frequency: 0.970 MHz; peak-rarefactional pressure: 0.52-MPa; acoustic power: 0.32 W; pulse repetition frequency: 1 Hz; duty cycle: 1–30%; duration: 3 min) on U87 MG cells, while DVDMS alone was non-toxic to the cells. In comparison with the control group, the SDT-treated group showed significant generation of intracellular ROS and loss of MMP at 1 h post-treatment. These results indicated that DVDMS-mediated SDT could induce great cytotoxicity in U87 MG cells via the production of ROS and showed potentials in the treatment for glioblastoma.

Terahertz confocal imaging: Polarization and sectioning characteristics

ABSTRACT The development of high-resolution THz imaging systems is currently of great practical significance, (e.g. in the areas of security, agri-food, biomedicine etc.). Implementing and calibrating such systems is essential, as is the verification and appropriate use of models. Confocal microscopes are well known to improve resolution and provide 3D images. In this paper a novel terahertz (THz) confocal transmission scanning microscopy is implemented using a commercial 300 GHz (~1 mm) electronic THz source and sensor array. In order to calibrate the system, two objects, a high contrast amplitude metallic object and a low contrast surface relief phase pattern, are produced. These simple samples are imaged to quantify the polarization and sectioning properties of the imaging system. A scalar model is shown to be appropriate and is shown to predict the measured lateral and axial resolutions. Experimental results using two more complex objects are then presented, confirming confocal operation. This paper details the implementation and calibration of a 3D THz confocal imaging system.

Gigapixel confocal imaging using a massively parallel optical probe array with single directional infinite scanning

Here we demonstrate high-throughput gigapixel confocal imaging using a massively parallel optical probe array with single directional infinite scanning. For implementation of the single directional infinite scan with high lateral resolution, a parallelogram array micro-objective lens module, composed of two wafer-level microlens arrays, is proposed to generate a massively parallel optical probe array for integration into the confocal imaging system, including an objective-side telecentric relay lens with a low-magnification. To test the feasibility of the proposed system with single directional infinite scanning, we designed and constructed a confocal imaging system using a parallelogram array of multi-optical probes with a massively parallel array size of 200 × 140. The constructed system provides a full width-half maximum lateral resolution of 1.55 μm, as measured by the knife-edge detection method, and a field-of-view width of 28.0 mm with a sampling interval of 1 μm/pixel.

Nonmydriatic widefield retinal imaging with an automatic white LED confocal imaging system compared with dilated ophthalmoscopy in screening for diabetic retinopathy.

To compare nonmydriatic montage widefield images with dilated fundus ophthalmoscopy for determining diabetic retinopathy (DR) severity. In this prospective, observational, cross-sectional study, patients with a previous diagnosis of diabetes and without history of diabetes-associated ocular disease were screened for DR. Montage widefield imaging was obtained with a system that combines confocal technology with white-light emitting diode (LED) illumination (DRSplus, Centervue, Padua, Italy). Dilated fundus examination was performed by a retina specialist. Thirty-seven eyes (20 patients, 8 females) were finally included in the analysis. Mean age of the patients enrolled was 58.0 ± 11.6years [range 31-80years]. The level of DR identified on montage widefield images agreed exactly with indirect ophthalmoscopy in 97.3% (36) of eyes and was within 1 step in 100% (37) of eyes. Cohen's kappa coefficient (κ) was 0.96, this suggesting an almost perfect agreement between the two modalities in DR screening. Nonmydriatic montage widefield imaging acquisition time was significantly shorter than that of dilated clinical examination (p = 0.010). Nonmydriatic montage widefield images were compared favorably with dilated fundus examination in defining DR severity; however, they are acquired more rapidly.