Imaging Chamber Research Articles

In-field grading and sorting technology of apples: A state-of-the-art review

Apple is one of the most popular fruits. In-field grading and sorting of apples would enhance growers’ economic benefits by lowering production costs. This study reviews the key components and progress of the quality inspection algorithm for in-field grading and sorting of apples. Four key components (e.g., conveyor, imaging chamber, sorting actuator, and bin filler) are presented in detail, followed by summarizing the shortcomings of these components. The apple’s external (color, size, and defects) and internal quality inspection technologies, such as optical technologies of visible light, near-infrared (NIR), hyperspectral/multispectral imaging (HSI/MSI), and structured illumination (SI) were presented. Despite the excellent detection performance of emerging technologies (e.g., HSI, MSI, and SI), visible light is still dominantly used for in-filed grading. Challenges in getting information on the whole surface area of the apple, uneven lighting, machine size, throughput, and associated costs hamper the commercialization of apple in-field grading and sorting equipment. At present, more efforts should be devoted to internal quality inspection, by developing reliable, fast, and accurate detection equipment and algorithms. With the advancement of sensors and automation algorithms, as well as the emergence of mechanical systems that are suitable for in-field use, it is anticipated that the apple in-field grading and sorting equipment that inspects both external and internal quality will be realized and commercialized in the near future.

RoPod, a customizable toolkit for non-invasive root imaging, reveals cell type-specific dynamics of plant autophagy

Arabidopsis root is a classic model system in plant cell and molecular biology. The sensitivity of plant roots to local environmental perturbation challenges data reproducibility and incentivizes further optimization of imaging and phenotyping tools. Here we present RoPod, an easy-to-use toolkit for low-stress live time-lapse imaging of Arabidopsis roots. RoPod comprises a dedicated protocol for plant cultivation and a customizable 3D-printed vessel with integrated microscopy-grade glass that serves simultaneously as a growth and imaging chamber. RoPod reduces impact of sample handling, preserves live samples for prolonged imaging sessions, and facilitates application of treatments during image acquisition. We describe a protocol for RoPods fabrication and provide illustrative application pipelines for monitoring root hair growth and autophagic activity. Furthermore, we showcase how the use of RoPods advanced our understanding of plant autophagy, a major catabolic pathway and a key player in plant fitness. Specifically, we obtained fine time resolution for autophagy response to commonly used chemical modulators of the pathway and revealed previously overlooked cell type-specific changes in the autophagy response. These results will aid a deeper understanding of the physiological role of autophagy and provide valuable guidelines for choosing sampling time during end-point assays currently employed in plant autophagy research.

NIRis: A low-cost, versatile imaging system for near-infrared fluorescence detection of phototrophic cell colonies used in research and education.

A variety of costly research-grade imaging devices are available for the detection of spectroscopic features. Here we present an affordable, open-source and versatile device, suitable for a range of applications. We provide the files to print the imaging chamber with commonly available 3D printers and instructions to assemble it with easily available hardware. The imager is suitable for rapid sample screening in research, as well as for educational purposes. We provide details and results for an already proven set-up which suits the needs of a research group and students interested in UV-induced near-infrared fluorescence detection of microbial colonies grown on Petri dishes. The fluorescence signal confirms the presence of bacteriochlorophyll a in aerobic anoxygenic phototrophic bacteria (AAPB). The imager allows for the rapid detection and subsequent isolation of AAPB colonies on Petri dishes with diverse environmental samples. To this date, 15 devices have been build and more than 7000 Petri dishes have been analyzed for AAPB, leading to over 1000 new AAPB isolates. Parts can be modified depending on needs and budget. The latest version with automated switches and double band pass filters costs around 350€ in materials and resolves bacterial colonies with diameters of 0.5 mm and larger. The low cost and modular build allow for the integration in high school classes to educate students on light properties, fluorescence and microbiology. Computer-aided design of 3D-printed parts and programming of the employed Raspberry Pi computer could be incorporated in computer sciences classes. Students have been also inspired to do agar art with microbes. The device is currently used in seven different high schools in Finland. Additionally, a science education network of Finnish universities has incorporated it in its program for high school students. Video guides have been produced to facilitate easy operation and accessibility of the device.

Integration of wide-field imaging system with droplet microfluidics for monitoring living bacteria

Droplet microfluidics has been widely used to analyze chemicals and biological reactions at the single-molecule level. Microscopic systems are commonly used for imaging and analyzing droplets using high-magnification objective lenses. However, these systems are expensive, complex, and large, limiting the high-throughput characterization of droplets in reactions targeting disease-related biomarkers, including nucleic acids, proteins, and pathogens. Another concern is the time gap between droplet analysis within a microfluidic channel and imaging chamber, which can cause discrepancies in reaction time between droplets. To address this issue, we introduce the droplet analysis system for vast imaging and statistical tool (DropVIST)—a wide-field imaging system integrated with droplet microfluidics for rapid and accurate droplet analysis. DropVIST can detect eight differently colored droplets simultaneously using a wide-field imaging system and dFinder software, which can quantify the reacted droplets. The smallest droplet diameter for detection was 30 μm, and the maximum detection area was 201.84 cm2, suggesting that the system can theoretically analyze 3103,377 droplets in real-time. We validated the monitoring performance of DropVIST using clinical isolates of five types of living pathogenic bacteria and compared this with conventional approaches, including the microbroth dilution method and colony-forming unit assay. This platform provides a rapid, simple, and accurate tool for monitoring living bacteria at the single-cell level.

Abstract 4149: Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models

Abstract Cancer immunotherapy utilizing the immunoreactive efficiency of immune cells via adoptive cell therapy (ACT) has been a novel method of cancer treatment. In tumor microenvironment, immune cell reactivity of tumor-infiltrating lymphocytes (TILs) can facilitate cancer cell attack, which finally derives efficient suppression of tumor growth. However, the conventional evaluation for cancer immunotherapy has been generally performed by ex vivo methods, providing restricted information on lymphocyte motility and immune cell dynamics within the tumor microenvironment. In vivo data with the dynamic behavior of TILs in breast cancer can provide new insight to investigate novel targets of immune checkpoint inhibitors. In this research, longitudinal intravital imaging of motility trafficking of TILs in breast cancer animal models has been performed by utilizing intravital microscopy to unveil the immunoreactive dynamics of T cell activity in early stage. In vivo analysis of TIL motility within different breast cell lines, including 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple-negative breast cancer cell), and MCF7 (human breast cancer cell), respectively, has been investigated for immune responses to cancer. We’ve monitored spatiotemporal dynamics of TIL over 11 days in breast cancer xenograft mouse model generated by tumor implantation into the dorsal skinfold imaging chamber and adoptive T cell transfer by blood circulation. In the results, the adoptive immunoreactivity of TIL has been differentiated by the timepoint of adoptive T cell transfer, distance proximity of T cells in tumors, and breast cancer types. Furthermore, anti-cancer effect coordinated with dendritic cells behaviors showed induced immune responses with accumulated dendritic cells with increased TIL count over time in breast cancer model treated with various cancer therapy such as immunotherapy, radiation therapy and anti-cancer drug (anti-PD-L1), which implied a relationship between programmed death-ligand 1 expression levels and TIL/dendritic cell motility. The result demonstrates that intravital TIL motility trafficking analysis in cancer model can be an invaluable assessment method to explain the detailed cellular/molecular mechanism of immune response to cancer involved with T lymphocytes, dendritic cells, and immune checkpoint inhibitors to accelerate the efficiency of cancer immunotherapy. Citation Format: Soyeon Ahn, Kubra Akyildiz, Hyunseok Kim, Pilhan Kim. Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4149.

Development of the multiplex imaging chamber atPAL-XFEL.

Various X-ray techniques are employed to investigate specimens in diverse fields. Generally, scattering and absorption/emission processes occur due to the interaction of X-rays with matter. The output signals from these processes contain structural information and the electronic structure of specimens, respectively. The combination of complementary X-ray techniques improves the understanding of complex systems holistically. In this context, we introduce a multiplex imaging instrument that can collect small-/wide-angle X-ray diffraction and X-ray emission spectra simultaneously to investigate morphological information with nanoscale resolution, crystal arrangement at the atomic scale and the electronic structure of specimens.

Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

Measurements of Methane Emissions from a Biofertilizer Storage Tank Using Ground-Based Hyperspectral Imaging and Flux Chambers.

Open storages of organic material represent potentially large sources of the greenhouse gas methane (CH4), an emissions source that will likely become more common as a part of societal efforts toward sustainability. Hence, monitoring and minimizing CH4 emissions from such facilities are key, but effective assessment of emissions without disturbing the flux is challenging. We demonstrate the capacity of using a novel high-resolution hyperspectral camera to perform sensitive CH4 flux assessments at such facilities, using as a test case a biofertilizer storage tank for residual material from a biogas plant. The camera and simultaneous conventional flux chamber measurements showed emissions of 6.0 ± 1.3 and 13 ± 5.7 kg of CH4 h-1, respectively. The camera measurements covered the whole tank surface of 1104 m2, and the chamber results were extrapolated from measurements over 5 m2. This corresponds to 0.7-1.4% of the total CH4 production at the biogas plant (1330 N m3 h-1 corresponding to 950 kg h-1). The camera could assess the entire tank emission in minutes without disturbing normal operations at the plant and revealed additional unknown emissions from the inlet to the tank (17 g of CH4 h-1) and during the loading of the biofertilizer into trucks (3.1 kg of CH4 h-1 during loading events). This study illustrates the importance of adequate measurement capacity to map methane fluxes and to verify that methane emission mitigation efforts are effective. Given the high methane emissions observed, it is important to reduce methane emissions from open storage of organic material, for example by improved digestion in the biogas reactor, precooling of sludge before storage, or building gastight storage tanks with sealed covers. We conclude that hyperspectral, ground-based remote sensing is a promising approach for greenhouse gas monitoring and mitigation.

Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest

BackgroundFor decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample’s morphology, thus making navigation to the ROI difficult.ResultsUsing the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems—charging and ROI localization—that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition.ConclusionsIn MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.

3D printed chamber for live cell imaging on an upright epifluorescence microscope

ABSTRACT Live cell imaging is a standard technique in experimental biology that enables the observation of isolated cells and tissue slices in real time and the testing of cellular responses to changes in buffer composition. However, most live cell imaging devices require the use of dedicated microscopes and/or specialised stage adaptors, and come at a reasonably high cost. We employed 3D printing technology to create a low-cost imaging chamber with side ports to exchange fluids, to be used on upright microscopes. The chamber increased the functionality of a standard upright epifluorescent microscope to allow dynamic, real-time calcium imaging of cultured hypothalamic astrocytes from mice, and to test the effects of ATP stimulation upon calcium signalling. It was also used on slices obtained from the mouse brain using a brain matrix slicer. The advantages of this chamber include a very simple design that can be used with upright epifluorescence microscopes, does not require any special stage adaptor, and includes ports to permit fluid exchange during imaging. This chamber is ideal for educational settings with undergraduate laboratories that do not have access to dedicated inverted fluorescent microscopes for tissue culture experiments.

Validating MALDI-IMS Feasibility in Ex Vivo Brain Slices.

Matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) generates unique mass spectra in X/Y coordinates across a tissue sample, thus allowing for the spatial detection and relative quantification of biologic compounds in situ. The soft ionization of MALDI-IMS makes it an ideal technique for high-resolution imaging of complex lipid species. Lipid-based spatial chemical maps derived from MALDI-IMS provide critical insight into the unique molecular profiles of a variety of neurologic diseases. Ex vivo brain slice preparations are a prominent alternative to in vivo animal models for studying many different neurologic conditions. For the first time, we present a feasible protocol for achieving reproducible lipidomic MALDI-IMS data from ex vivo rat brain slices and provide evidence that ex vivo brain slices maintain spatiochemical lipidomic profiles representative of an intact whole brain. We conducted a methods comparison assessing the lipid profiles within the neocortex, striatum, and corpus callosum between coronal sections taken from ex vivo brain slices and the current gold standard tissue preparation method, fresh frozen whole brains. For the first time we demonstrate a technique by which 400 μm ex vivo brain slices can be extracted from an imaging chamber and prepared for MALDI-IMS in a way that preserves their lipidomic integrity. We demonstrate the feasibility of MALDI-IMS in ex vivo brain slices and provide a roadmap for MALDI-IMS utilization in uncharted neuroscience fields.

A Quad-Band Near-Field Antenna Array for a Multistatic Microwave Imaging Chamber

A new quad-band tapered patch transmit-receive antenna array designed for a multistatic microwave imaging cavity system is presented. The reverberating cube-shaped chamber is filled with a matching fluid emulsion and encompasses 64 antenna elements mounted on its sidewalls (16 per panel). Four resonant frequencies that lie in the range of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$0.5 - 3$</tex-math></inline-formula> GHz are achieved. The design procedure is provided based on system constraints and performance requirements that need to be taken into account: (1) the near-field nature of the electromagnetic wave in the presence of a shielded chamber and (2) the propagation of waves into the immersion fluid within the imaging domain. The antenna array design was numerically verified, and its prototype was fabricated. Results show good agreement between measurements and simulations, achieving antenna system performance requirements.

Gold Ions Hyperpolarize Bacteria

Background: The study of bacterial electrophysiology is important for understanding antibacterial resistance, biofilm formation, and fundamental questions of cell growth and division. These experiments require new tools to modulate the resting membrane potential of bacteria. The use of potassium and magnesium ions, antibiotics, short exposures to blue light, and electric field have been used to hyperpolarize or depolarize bacteria. Our research characterizes the cellular response to a new reagent, gold ions, using single-cell imaging. Methods: We use fluorescence microscopy to monitor, in real time, the change to the membrane potential when Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli) are exposed to gold ions. Membrane potential is measured with two different Nernstian dyes, thioflavin T and tetramethylrhodamine methyl ester. Gold ions are introduced to cell by adding gold salt solutions or through the electrochemical generation of gold ions. Inductively coupled plasma mass spectrometry (ICP-MS) is used to determine the concentration of gold ions generated electrochemically. Standard growth curves are used to study the effect of gold ions on bacterial growth. Results: Single cell fluorescence imaging shows that a solution of gold ions hyperpolarizes B. subtilis and E. coli in a concentration-dependent manner. ICP-MS confirms that applied voltage and frequency controls the concentration of gold ions generated electrochemically. Electrochemically generated gold ions diffuse through the imaging chamber creating a wave of hyperpolarization. The speed of the hyperpolarization wave can be modulated by voltage and frequency. Conclusions: Our research describes a new tool, gold ions, for the hyperpolarization of bacteria. Gold ions can be introduced to bacteria as a salt solution or generated electrochemically, providing spatial and temporal control of bacterial membrane potential. Beyond this work, it is possible that hyperpolarization may play a role in the use of gold as an antibacterial agent.

Comparing dietary intake of free-feeding to feeding in restrained imaging chamber environments using seven days post fertilization larvae.

Heart disease and diabetes are both in the top ten for leading causes of death in the United States with both being linked to obesity. Studying human metabolism can prove difficult due to ethical concerns with human subjects but zebrafish larvae (Danio rerio) are optically transparent. Zebrafish larvae at seven days post fertilization also have fully formed digestive tracts and are actively seeking new food due to exhausting their supply of egg yolk. Free feeding environments are typically the standard when introducing new food to the larvae but lack versatility in their imaging. The Walters lab microfluidic chip can image larvae before, during, and after their introduction to the diet. To prove the two types of feeding environments were compared by using fluorescent digestive markers mixed into high fat diets. The free fed larvae were put into a well filled with diet and allowed to feed for three hours while sat on a orbital shaker inside of a 28 degrees Celsius incubator. After three hours, these larvae were rinsed out and imaged. The microfluidic chip larvae were mounted into the chip and fed the diet while inside and under the same Zeiss Discovery V8 microscope the freely fed larvae were imaged under. Future predictions for this data were that the larvae would consume slightly less diet inside of the microfluidic chip than the larvae in free feed. Results for this are still ongoing but seem to lead towards supporting the hypothesis.

Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy.

Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid-phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high-vacuum (10-6 Pa) and low-vacuum (80-120Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy.

A method for reproducible high-resolution imaging of 3D cancer cell spheroids.

Multicellular tumour cell spheroids embedded within three-dimensional (3D) hydrogels or extracellular matrices (ECM) are widely used as models to study cancer growth and invasion. Standard methods to embed spheroids in 3D matrices result in random placement in space which limits the use of inverted fluorescence microscopy techniques, and thus the resolution that can be achieved to image molecular detail within the intact spheroid. Here, we leverage UV photolithography to microfabricate PDMS (polydimethylsiloxane) stamps that allow for generation of high-content, reproducible well-like structures in multiple different imaging chambers. Addition of multicellular tumour spheroids into stamped collagen structures allows for precise positioning of spheroids in 3D space for reproducible high-/super-resolution imaging. Embedded spheroids can be imaged live or fixed and are amenable to immunostaining, allowing for greater flexibility of experimental approaches. We describe the use of these spheroid imaging chambers to analyse cell invasion, cell-ECM interaction, ECM alignment, force-dependent intracellular protein dynamics and extension of fine actin-based protrusions with a variety of commonly used inverted microscope platforms. This method enables reproducible, high-/super-resolution live imaging of multiple tumour spheroids, that can be potentially extended to visualise organoids and other more complex 3D in vitro systems.

A Novel Window into Angiogenesis-Intravital Microscopy in the AV-Loop-Model.

Due to the limitations of current in vivo experimental designs, our comprehensive knowledge of vascular development and its implications for the development of large-scale engineered tissue constructs is very limited. Therefore, the purpose of this study was to develop unique in vivo imaging chambers that allow the live visualization of cellular processes in the arteriovenous (AV) loop model in rats. We have developed two different types of chambers. Chamber A is installed in the skin using the purse sting fixing method, while chamber B is installed subcutaneously under the skin. Both chambers are filled with modified gelatin hydrogel as a matrix. Intravital microscopy (IVM) was performed after the injection of fluorescein isothiocyanate (FITC)-labeled dextran and rhodamine 6G dye. The AV loop was functional for two weeks in chamber A and allowed visualization of the leukocyte trafficking. In chamber B, microvascular development in the AV loop could be examined for 21 days. Quantification of the microvascular outgrowth was performed using Fiji-ImageJ. Overall, by combining these two IVM chambers, we can comprehensively understand vascular development in the AV loop tissue engineering model¯.

Noninvasive Long-Term Imaging of the Cytoskeleton in Arabidopsis Seedlings.

The preparation of biological samples, especially for live-cell microscopy, remains a major experimental challenge in the lab despite technological advances. In addition, high-resolution microscopy techniques require higher sample quality and uniformity, which is difficult to ensure during manual preparation while maintaining "ideal" growth conditions. In this protocol, we provide a way out by growing Arabidopsis thaliana seedlings directly in an imaging chamber, which eliminates invasive sample preparation directly before imaging. This method hinges on the precise placement of seeds into imaging chambers, which can be grown in conventional climate chambers. We detail three methods to grow hypocotyls, cotyledons, leaves, and roots for high-resolution and long-term imaging of the plant cytoskeleton. Furthermore, we show that the growth and development of seedlings inside the chambers can be externally manipulated by the addition of chemicals.

Effects of valproate on seizure-like activity in Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations as a model of Angelman Syndrome

Angelman Syndrome is a rare, genetically induced neurodevelopmental disorder. This disorder stems from a mutation or deletion of the maternal UBE3A gene. Characteristics of this disease include developmental delay, recurring seizures, and severe intellectual disabilities. We studied seizure activity in male Drosophila melanogaster with a knockdown of Ube3a in different neuronal populations (GABAergic, glutamatergic, mushroom body, and all neurons) and investigated the effects of the antiseizure medication (ASM) on seizure-like activity. Epileptiform activity was monitored in individual fruit flies using imaging chambers and mechanically induced seizures using a vortex assay. A positive control was also used: eas (easily shocked seizure phenotype). Seizure activity was analyzed for sums of seizure durations, number of seizures, and total time to return to normal activity.Ube3a knockdowns in GABAergic neurons elicited more seizure-like episodes than knockdowns in glutamatergic neurons and were on par with the positive control group and those with knockdowns in the mushroom bodies. We have established a method whereby valproate could be administered through food rather than through injections to effectively treat epileptiform activity. We demonstrated that if Ube3a is not knocked down pan-neuronally, Angelman Syndrome seizure-like activity can be studied using Drosophila melanogaster and therefore allows for high-throughput drug discovery.