assembly-operation-cooling-stage-to-immobilize-c-elegans-on-their

Assembly and Operation of a Cooling Stage to Immobilize <em>C. elegans</em> 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), ...

Northeastern University

Multichannel (multicolor) imaging has become a powerful technique in biology research for performing in vivo neuronal calcium imaging, colocalization of fluorescent labels, non-invasive pH measurement, and other procedures. We describe a novel add-on approach for simultaneous multichannel optical microscopy based on simple wedge prisms. Our device requires no alignment and is simple, robust, user-friendly, and less expensive than current commercial instruments based on switchable filters or dual-view strategies. Point spread function measurements and simulations in Zemax indicate a reduction in resolution in the direction orthogonal to the wedge interface and in the axial direction, without introducing aberration. These effects depend on the objective utilized and are most significant near the periphery of the field of view. We tested a two-channel device on C. elegans neurons in vivo and demonstrated comparable signals to a conventional dual-view instrument. We also tested a four-channel device on fixed chick embryo Brainbow samples and identified individual neurons by their spectra without extensive image postprocessing. Therefore, we believe that this technology has the potential for broad use in microscopy.

Wedge prism approach for simultaneous multichannel microscopy.

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 at 1.4 NA 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 μm for our SLM-enhanced setup and 0.68 ± 0.04 μm 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 96% of the dim neuronal fibers seen in confocal images while a widefield microscope only identified 50% 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.

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

Femtosecond lasers are capable of precise ablation that produces surgical dissections in vivo. The transverse and axial resolutions 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. Using a 1040-nm, 400-fs pulsed laser, and a 1.4-NA objective, 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 Caenorhabditis 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.

Transverse and axial resolution of femtosecond laser ablation.

Despite its profound impact on biology, high-resolution  microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode  directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging 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. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows.

Article	Year
Wedge prism approach for simultaneous multichannel microscopy. Scientific reports\| PubMed ID: 31780755	2019
Confocal imaging capacity on a widefield microscope using a spatial light modulator. PloS one\| PubMed ID: 33591984	2021
Transverse and axial resolution of femtosecond laser ablation. Journal of biophotonics\| PubMed ID: 35583201	2022
High-throughput submicron-resolution microscopy of populations under strong immobilization by cooling cultivation plates. iScience\| PubMed ID: 36794150	2023

Yao L. Wang

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