Biology subjects

Grooms, N. W. F.

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

3 recordsLinked to original sources

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↗