Biology subjects

Grooms, N. W. F.

Publications and source records attributed to Grooms, N. W. F..

5 recordsLinked to original sources

bioRxiv2024

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-{varepsilon}/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

cell biology↗

bioRxiv2023

Assembly and operation of a cooling stage to immobilize C. elegans on their culture plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans) but requires strong animal immobilization. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. We greatly simplify the C. elegans immobilization procedure by using a cooling approach that can easily immobilize entire populations of C. elegans directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures on the cultivation plate and ensures even temperature distribution across the agarose surface. In this article, we document the whole process of building the cooling stage from scratch. We envision that a typical researcher can build an operational cooling stage in their lab following our protocol without difficulty. We show how to utilize the cooling stage under three protocols, which have advantages for different experiments. We also show an example cooling profile of the stage as it approaches its final temperature and include some helpful tips in using cooling immobilization.

bioengineering↗

bioRxiv2022

Transverse and axial resolution of femtosecond laser ablation

Femtosecond lasers are capable of precise ablation that produce surgical dissections in vivo. The transverse and axial resolution of the laser damage inside the bulk are important parameters of ablation. The transverse resolution is routinely quantified, but the axial resolution is more difficult to measure and is less commonly performed. In some in vivo samples, fine dissections can also be difficult to visualize, but in vitro samples may allow clear imaging. Using a 1040-nm, 400-fs pulsed laser, we performed ablation inside agarose and glass, producing clear and persistent damage spots. Near the ablation threshold of both media, we found that the axial resolution is similar to the transverse resolution. We also ablated neuron cell bodies and fibers in C. elegans and demonstrate submicrometer resolution in both the transverse and axial directions, consistent with our results in agarose and glass. Using simple yet rigorous methods, we define the resolution of laser ablation in transparent media along all directions.

bioengineering↗

bioRxiv2021

Immobilization of C. elegans on cultivation plates by thermoelectric cooling for high-throughput subcellular-resolution microscopy

Despite its profound impact on biology, most high-resolution in vivo microscopy approaches remain low throughput because current immobilization techniques require significant manual effort. We greatly accelerate imaging of the nematode Caenorhabditis elegans by implementing a simple cooling approach to easily immobilize entire populations directly on their cultivation plates. We optimize and characterize cooling immobilization. Counterintuitively, relatively warmer temperatures immobilize animals significantly more effectively than colder temperatures utilized in prior studies. This enhanced immobilization enables clear submicron-resolution fluorescence imaging, which is challenging to achieve with most current immobilization techniques. We demonstrate 64x magnification 3D imaging and timelapse recording of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. By obviating individual animal manipulation, our approach could also empower automated imaging of large C. elegans populations within standard experimental setups and workflows.

bioengineering↗

bioRxiv2020

Confocal imaging capacity on a widefield microscope using a spatial light modulator

Confocal microscopes can reject out-of-focus and scattered light; however, widefield microscopes are far more common in biological laboratories due to their accessibility and lower cost. We report confocal imaging capacity on a widefield microscope by adding a spatial light modulator (SLM) and utilizing custom illumination and acquisition methods. We discuss our illumination strategy and compare several procedures for postprocessing the acquired image data. We assessed the performance of this system for rejecting out-of-focus light by comparing images taken using our widefield microscope, our SLM-enhanced setup, and a commercial confocal microscope. The optical sectioning capability, assessed on thin fluorescent film, was 0.85 {+/-} 0.04 m for our SLM-enhanced setup and 0.68 {+/-} 0.04 m for a confocal microscope, while a widefield microscope exhibited no sectioning capability. We demonstrate our setup by imaging the same set of neurons in C. elegans on widefield, SLM, and confocal microscopes. SLM enhancement greatly reduces background from the cell body, allowing visualization of dim fibers nearby. Our SLM-enhanced setup identified 93% of the dim neuronal fibers seen in confocal images while a widefield microscope only identified 48% of the same fibers. Our microscope add-on represents a very simple (2-component) and inexpensive (<$600) approach to enable widefield microscopes to optically section thick samples.

bioengineering↗