Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing.

Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.

In vivo hamster cheek pouch subepithelial ablation, biomaterial injection, and localization: pilot study.

SIGNIFICANCE: The creation of subepithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable biomaterials toward a clinically viable therapy for vocal fold scarring. AIM: We aim to prove that subepithelial voids can be created in a live animal model and that the ablation process does not engender additional scar formation. We demonstrate localization and long-term retention of an injectable biomaterial within subepithelial voids. APPROACH: A benchtop nonlinear microscope was used to create subepithelial voids within healthy and scarred cheek pouches of four Syrian hamsters. A model biomaterial, polyethylene glycol tagged with rhodamine dye, was then injected into these voids using a custom injection setup. Follow-up imaging studies at 1- and 2-week time points were performed using the same benchtop nonlinear microscope. Subsequent histology assessed void morphology and biomaterial retention. RESULTS: Focused ultrashort pulses can be used to create large subepithelial voids in vivo. Our analysis suggests that the ablation process does not introduce any scar formation. Moreover, these studies indicate localization, and, more importantly, long-term retention of the model biomaterial injected into these voids. Both nonlinear microscopy and histological examination indicate the presence of biomaterial-filled voids in healthy and scarred cheek pouches 2 weeks postoperation. CONCLUSIONS: We successfully demonstrated subepithelial void formation, biomaterial injection, and biomaterial retention in a live animal model. This pilot study is an important step toward clinical acceptance of a new type of therapy for vocal fold scarring. Future long-term studies on large animals will utilize a miniaturized surgical probe to further assess the clinical viability of such a therapy.

Planar Laser Activated Neuronal Scanning (PLANS) System for in vivo Flow Cytometry.

We present a light-sheet flow cytometer for screening of C. elegans. A machine learning approach is utilized to enable real-time analysis of protein aggregation models.

Variation in CT Number and Image Noise Uniformity According to Patient Positioning in MDCT.

OBJECTIVE: Many algorithms for clinical decision making rely on assessment of the CT number (expressed as Hounsfield units); however, to our knowledge, few, if any, studies have addressed how CT numbers change as a function of patient positioning within the scanner. MATERIALS AND METHODS: An anthropomorphic phantom underwent imaging with varying amounts of vertical orientation misalignment with respect to isocenter. CT number and noise were measured using ROIs in the upper thorax, mid thorax, and abdomen. The degree of noise nonuniformity and changes in the CT number were assessed by comparing values obtained in the anterior versus posterior ROIs. To add clinical relevance, data on vertical mispositioning were collected from 20,316 clinical abdominal CT scans. Box-and-whisker plot analysis was used to identify the range of patient positioning. RESULTS: Absolute CT number changes of more than 20 HU were observed for some ROIs at phantom positions of 10 cm from isocenter, with important differences noted between the thoracic and abdominal regions. Noise uniformity varied by more than twofold for all regions at 10 cm below isocenter. On clinical CT examinations, off-centering of more than 1, 2, 4, and 6 cm occurred for 41%, 19%, 1.9%, and 0.3% of patients, respectively. CONCLUSION: Radiologists should treat CT number measurements with caution when patients are grossly mispositioned in the scanner. The substantial changes in attenuation values shown in the present study are large enough to warrant further investigation.

A Wiki Based CT Protocol Management System.

At the University of Wisconsin Madison Department of Radiology, CT protocol management requires maintenance of thousands of parameters for each scanner. Managing CT protocols is further complicated by the unique configurability of each scanner. Due to recent Joint Commission requirements, now all CT protocol changes must be documented and reviewed by a site's CT protocol optimization team. The difficulty of managing the CT protocols was not in assembling the protocols, but in managing and implementing changes. This is why a wiki based solution for protocol management was implemented. A wiki inherently keeps track of all changes, logging who made the changes and when, allowing for editing and viewing permissions to be controlled, as well as allowing protocol changes to be instantly relayed to all scanner locations.