Affordable Diffusion Microchamber Array Designs for Isolating Microbes in Classrooms and Laboratories.

Diffusion microchamber array (DMA) devices have provided contemporary microbiologists with a successful alternative to the century-old plating method using petri dishes to isolate and cultivate environmental bacteria. The "ichip" DMA device, developed by the research group of Slava Epstein, is exemplary of this technology, using membranes to culture rare or otherwise nonmodel bacterial taxa, whereby the target cells are nourished by environmental medium across the separating membrane. However, DMA devices have not sufficiently infiltrated the university-level microbiology curriculum, and the size and shape of the ichip make the device challenging for use by students. The following work provides guidance in the creation and use of a suite of DMA devices called "iplates," which are ichip-like tools for use in microbiology education. An iplate mimics the shape and size of a 96-well plate, making it a simpler, more affordable, and customizable design for the isolation and incubation of bacteria and other microorganisms using fresh environmental media. Iplates and their many customizations are intended to enable teachers, students, and researchers to isolate, grow, and analyze the widest possible diversity of microorganisms, complementing standard plating methods. We offer additional tips and tools to help instructors and students get started creating DMA devices to meet their unique needs and research interests.

Open Source Remote Monitoring of Research Lasers.

An open source remote monitoring system is designed and built to address the needs of researchers to provide basic illuminated visual indication of laser operation for university research laboratories that are equipped with multiple types of high-powered lasers and have limited financial resources. The 3D printed remote monitoring system selectively monitors either the total current running through a laser or a TTL shutter signal to wirelessly indicate at the laboratory entrances that a laser is in use. Several lasers can be monitored in a single room and each room entrance can have its own wireless laser activity indicator. The wireless feature eliminates the expense of in-wall wiring for the system. An emergency shut off switch is included as an optional attachment. This article describes the design of the readily deployed open source laser monitoring system, including how it was built and tested for integration into a microscopy research laboratory.

MRI Compatible, Customizable, and 3D-Printable Microdrive for Neuroscience Research.

The effective connectivity of brain networks can be assessed using functional magnetic resonance imaging (fMRI) to quantify the effects of local electrical microstimulation (EM) on distributed neuronal activity. The delivery of EM to specific brain regions, particularly with layer specificity, requires MRI compatible equipment that provides fine control of a stimulating electrode's position within the brain while minimizing imaging artifacts. To this end, we developed a microdrive made entirely of MRI compatible materials. The microdrive uses an integrated penetration grid to guide electrodes and relies on a microdrilling technique to eliminate the need for large craniotomies, further reducing implant maintenance and image distortions. The penetration grid additionally serves as a built-in MRI marker, providing a visible fiducial reference for estimating probe trajectories. Following the initial implant procedure, these features allow for multiple electrodes to be inserted, removed, and repositioned with minimal effort, using a screw-type actuator. To validate the design of the microdrive, we conducted an EM-coupled fMRI study with a male macaque monkey. The results verified that the microdrive can be used to deliver EM during MRI procedures with minimal imaging artifacts, even within a 7 Tesla (7T) environment. Future applications of the microdrive include neuronal recordings and targeted drug delivery. We provide computer aided design (CAD) templates and a parts list for modifying and fabricating the microdrive for specific research needs. These designs provide a convenient, cost-effective approach to fabricating MRI compatible microdrives for neuroscience research.

A syringe adapter for reduced muscular strain and fatigue.

Workers in hospitals, clinics, and contract research organizations who repetitively use syringes have an increased risk for musculoskeletal disorders. This study developed and tested a novel syringe adapter designed to reduce muscle strain associated with repetitive fluid draws. Three syringe plunger extension methods (ring-finger, middle-finger, and syringe adapter) were studied across twenty participants. Electromyogram signals for the flexor digitorum superficialis and extensor digitorum muscles were recorded. The syringe adapter required 31% of the 90th percentile flexor muscle activity as compared to the ring-finger syringe extension method, and 45% the 90th percentile flexor muscle activity as compared to the middle-finger method (p < 0.001). The greatest differences were observed when the syringe was near full extension. Although the syringe adapter took more time than the other syringe extension methods (1.5 times greater), it greatly helped reduce physical stress associated with repetitive, awkward syringe procedures.

A novel bioreactor for combined magnetic resonance spectroscopy and optical imaging of metabolism in 3D cell cultures.

PURPOSE: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized (DNP) 13 C-pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures. METHODS: The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI-compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three-dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts. RESULTS: Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized 13 C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose-deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ1 (free NADH) showed no significant change, while τ2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH. CONCLUSION: A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and 13 C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied.

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

3D second harmonic generation imaging tomography by multi-view excitation.

Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.

zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.

Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.