Ultrasound, photoacoustic, and magnetic resonance imaging to study hyperacute pathophysiology of traumatic and vascular brain injury.

BACKGROUND AND PURPOSE: Cerebrovascular dynamics and pathomechanisms that evolve in the minutes and hours following traumatic vascular injury in the brain remain largely unknown. We investigated the pathophysiology evolution in mice within the first 3 hours after closed-head traumatic brain injury (TBI) and subarachnoid hemorrhage (SAH), two significant traumatic vascular injuries. METHODS: We took a multimodal imaging approach using photoacoustic imaging, color Doppler ultrasound, and MRI to track injury outcomes using a variety of metrics. RESULTS: Brain oxygenation and velocity-weighted volume of blood flow (VVF) values significantly decreased from baseline to 15 minutes after both TBI and SAH. TBI resulted in 19.2% and 41.0% ipsilateral oxygenation and VVF reductions 15 minutes postinjury, while SAH resulted in 43.9% and 85.0% ipsilateral oxygenation and VVF reduction (p < .001). We found partial recovery of oxygenation from 15 minutes to 3 hours after injury for TBI but not SAH. Hemorrhage, edema, reduced perfusion, and altered diffusivity were evident from MRI scans acquired 90-150 minutes after injury in both injury models, although the spatial distribution was mostly focal for TBI and diffuse for SAH. CONCLUSIONS: The results reveal that the cerebral oxygenation deficits immediately following injuries are reversible for TBI and irreversible for SAH. Our findings can inform future studies on mitigating these early responses to improve long-term recovery.

Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification.

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis.

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.

Smartphone-Based Paper Microfluidic Immunoassay of Salmonella and E. coli.

Previous studies from our lab have created a simple procedure for single-cell count of bacteria on a paper chip platform using optical detection from a smartphone. The procedure and steps employed are outlined along with the lessons learned and details of certain steps and how the design was optimized. Smartphone optical detection is easy to use, low cost, and potentially field deployable, which can be useful for early and rapid detection of pathogens. Smartphone imaging of a paper microfluidic chip preloaded with antibody-conjugated particles provides an adaptable platform for detection of different bacterial targets. The paper microfluidic chip was fabricated with a multichannel design. Each channel was preloaded with either a negative control of bovine serum albumin (BSA) conjugated particles, anti-Salmonella Typhimurium-conjugated particles with varying amounts (to cover different ranges of assay), or anti-Escherichia coli-conjugated particles. Samples were introduced to the paper microfluidic chip using pipetting. Antigens of Salmonella Typhimurium traveled through the channel by capillary action confined within the paper fibers surrounded by the hydrophobic barrier. The paper channel was observed to act as a filter for unwanted particles and contaminants found in field samples. Serial dilutions of known concentrations of bacterial targets were also tested using this procedure to construct a standard curve prior to the assays. The antibody-conjugated particles were able to immunoagglutinate which was quantified through evaluation of Mie scatter intensity. This Mie scattering was quantified in images taken with a smartphone at an optimized angle and distance. Mie scatter simulation provided a method of optimizing the experimental setup and could translate easily to other types of target sample matrices. A smartphone application was developed to help the user position the smartphone optimally in relation to the paper microfluidic chip. The application integrated both image capturing capability and a simple image processing algorithm that calculated bacteria concentrations. The detection limit was at a single-cell level with a total assay time ranging from 90 to less than 60 s depending on the target.