[
International Worm Meeting,
2021]
A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. Lesion conditioning could be utilized to drive powerful therapies for neuroinjuries. Despite being studied for >30 years, lesion conditioned regeneration remains poorly understood, and progress is severely limited by low throughput in vertebrate models. To expedite research in the field, we are developing a model for lesion-conditioned regeneration in C. elegans. Our model employs green fluorescent protein (GFP) to label the ASJ neuron. When we condition the neuron, we see increased fluorescence in the ASJ, indicating a correlation between GFP expression and regenerative capacity. Meanwhile, following prior work from our laboratory, disruptions to the sensory pathway can also chronically condition the neuron, increasing regenerative capacity without the need of a conditioning lesion. We saw increased fluorescence in chronically conditioned strains, further supporting the correlation between GFP expression and regenerative capacity. We used ethyl methanesulfonate to stochastically introduce mutations into a chronically conditioned strain and selected for offspring with decreased ASJ fluorescence, indicating a mutation in a gene potentially in the conditioning pathway. We isolated twelve strains, originating from six distinct F1s. Six of these strains show reduced frequency of ectopic axon outgrowths compared to the pre-mutagenized strain. A reduction in ectopic outgrowths suggests a disruption in the conditioning pathway, as previously characterized by our laboratory. To quantify the fluorescent correlation, we imaged the mutagenized strains with calibrated fluorescent beads and compared fluorescent intensities of the ASJ neurons to strains with increased outgrowths and regenerative potential. We found significantly decreased brightness in cell bodies and dendrites in strains with reduced regenerative potential compared to chronically conditioned strains. This correlation provides a powerful proxy for evaluating regenerative capacity and to identify genes potentially implicated in the lesion conditioned pathway.
[
International Worm Meeting,
2021]
Widefield microscopes are commonly used in many biological laboratories. However, their inability to reject scattered or out-of-focus light often produces images with obscured thin or dim structures when there are some bright structures nearby. Scanning microscopes like confocal microscopes and multiphoton microscopes can solve this problem, but they are comparatively slower and much more expensive. We developed an inexpensive way to empower confocal imaging capacity on a widefield microscope, by inserting a spatial light modulator (SLM) into the field stop of the widefield microscope and customizing the illumination pattern and acquisition methods. We assessed the performance of this SLM-inserted setup by comparing images taken at our widefield microscope, our widefield microscope with the SLM-inserted setup, and a commercial confocal microscope. While a widefield microscope showed no sectioning capability, our SLM-inserted setup showed 0.85 ± 0.04 mum and the commercial confocal showed 0.68 ± 0.04 mum optical sectioning capability. Additionally comparing images of the FLP neuron and the tightly bundled amphid neurons in C. elegans taken by the widefield, SLM-inserted setup, and confocal microscopes, we confirmed that the SLM-inserted setup greatly reduces haze from the bright cell body, allowing visualization of dim axons and dendrites nearby. Our SLM-inserted setup identified 96% of the dim neuronal fibers seen in confocal images while the widefield microscope only identified 50% of the same fibers. Our SLM-inserted setup represents a very simple (2-component) and inexpensive (<$600) approach to enable confocal capacity on a widefield microscope. This SLM-inserted setup can be broadly employed by labs that are using widefield microscopes, with minimum expense and modification.
[
International Worm Meeting,
2021]
Microscopy, surgical techniques, and imaging in vivo are broadly utilized for model organisms. However, despite widespread usage, these strategies for multicellular organisms remain low-throughput and require significant manual involvement. Here, we report the implementation of a novel cooling stage to immobilize Caenorhabditis elegans on typical agar cultivation plates for these purposes. This device can effectively cool C. elegans to between 1-2 degrees Celsius for immobilization and maintain the temperature with minimal fluctuations. We demonstrate the ability to perform imaging and surgical techniques without classic anesthetic agents like sodium azide. This technique decreases animal processing time while maintaining organism viability and fecundity, using an intuitive device built with attainable materials. Our thermoelectric cooling stage, which is highly effective and built to combine with standard microscopy setups, can enable high-throughput microscopy and surgical techniques with decreased manual and chemical interventions.