High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution

Rapid analysis of organic microcontaminants in environmental water samples by trace enrichment and liquid chromatography on a single short column.

On-column trace enrichment and liquid chromatography using a single short (20 mm length) high-pressure packed column was optimized for the rapid simultaneous identification and quantification of a wide range of organic microcontaminants in environmental water samples. The quality of different C,, bonded silica packing materials in terms of their performance (i.e., peak capacity, repeatability) and enrichment/extraction characteristics (i.e., recovery, selectivity) was tested. With 15-ml samples, limits of detection of 0.1-0.4 μg/l and 0.5-1.1 μg/l were achieved for tap and surface water, respectively. The applicability of the system was demonstrated by the screening of real environmental samples and the provisional identification of unknowns

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized

Demanding and effective : participants' experiences of internet-delivered prolonged exposure provided within two months after exposure to trauma

Background: The use of remotely delivered early intervention after trauma may prevent and/or reduce symptoms of post-traumatic stress. Our research group evaluated a novel three-week therapist-guided internet-delivered intervention based on prolonged exposure (Condensed Internet-Delivered Prolonged Exposure; CIPE) in a pilot trial. The results indicated that the intervention was feasible, acceptable and reduced symptoms of post-traumatic stress at post-intervention compared to a waiting-list condition. Exposure to traumatic memories can be emotionally demanding and there is a need for detailed investigation of participants? experiences in receiving this type of intervention remotely. Objective: Investigate participants? experiences of receiving CIPE early after trauma.Method: In this study, qualitative thematic analysis was used and semi-structured interviews with 11 participants six months after intervention completion were conducted. All interviews were audio-recorded and transcribed verbatim. Results: One overarching theme labelled as ?demanding and effective? was identified. Participants expressed that treatment effects could only be achieved by putting in a lot of effort and by being emotionally close to the trauma memory during exposure exercises. Participants reported CIPE to be a highly credible- and educative intervention that motivated them to fully engage in exposure exercises. The most distressing parts of the intervention was perceived as tolerable and important to do to heal psychologically after trauma. For many participants, the possibility to engage in the intervention whenever and where it suited them was helpful, although some participants described it as challenging to find a balance between their own responsibility and when to expect therapist support. The internet-based format was perceived as a safe forum for self-disclosure that helped some participants overcome avoidance due to shame during imaginal exposure. Conclusion: CIPE was considered demanding, yet effective by the interviewed participants. The most distressing parts of the intervention was perceived to be the most important and were tolerable and feasible to provide online