Elegans Larvae Research Articles

Impact of photobleaching on quantitative, spatio-temporal, super-resolution imaging of mitochondria in live C. elegans larvae

Super-resolution (SR) 3D rendering allows superior quantitative analysis of intracellular structures but has largely been limited to fixed or ex vivo samples. Here we developed a method to perform SR live imaging of mitochondria during post-embryonic development of C. elegans larvae. Our workflow includes the drug-free mechanical immobilisation of animals using polystyrene nanobeads, which has previously not been used for in vivo SR imaging. Based on the alignment of moving objects and global threshold-based image segmentation, our method enables an efficient 3D reconstruction of individual mitochondria. We demonstrate for the first time that the frequency distribution of fluorescence intensities is not affected by photobleaching, and that global thresholding alone enables the quantitative comparison of mitochondria along timeseries. Our composite approach significantly improves the study of biological structures and processes in SR during C. elegans post-embryonic development. Furthermore, the discovery that image segmentation does not require any prior correction against photobleaching, a fundamental problem in fluorescence microscopy, will impact experimental strategies aimed at quantitatively studying the dynamics of organelles and other intracellular compartments in any biological system.

3D manipulation of small multicellular organisms in soft microtubes

Small multicellular organisms, such as C. elegans and zebrafish larvae, are essential models in biological research, but their 3D manipulation poses challenges due to their small size. Manual positioning is labor-intensive and imprecise. To address this, we developed a soft PDMS microtube that can be gently stretched and released, immobilizing organisms without anesthetization and damage. This tube enables easy rotation and adjustment of orientation, facilitating comprehensive imaging. Functionality testing showed effective immobilization as well as precise imaging and microinjection. Zebrafish larvae were successfully injected with fluorescein in the hindbrain without anesthesia. This technique offers a simple, efficient, and non-damaging method for 3D manipulation and imaging of small multicellular organisms and provides a versatile tool for biological research practices.

Cryo-electron tomography elucidates annular intraluminal configurations in Caenorhabditis elegans microtubules.

Microtubules serve as integral components in cellular operations such as cell division, intracellular trafficking, and cellular architecture. Composed of tubulin protein subunits, these hollow tubular structures have been increasingly elucidated through advanced cryo-electron microscopy (Cryo-EM), which has unveiled the presence of microtubule inner proteins (MIPs) within the microtubular lumen. In the present investigation, we employ a synergistic approach incorporating high-pressure freezing, cryo-focused ion beam milling, and Cryo-electron tomography (Cryo-ET) to interrogate the in situ architecture of microtubules in Caenorhabditis elegans larvae. Our Cryo-ET assessments across neuronal cilia and diverse tissue types consistently demonstrate the formation of annular configurations within the microtubular lumen. In concert with recently characterized MIPs, our in situ observations within a living organism corroborate the hypothesis that intricate luminal assemblages exist within microtubule scaffolds. These findings necessitate further exploration into the molecular constituents and functional ramifications of these internal microtubular configurations in both cellular physiology and pathophysiology.

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Anchorage of H3K9-methylated heterochromatin to the nuclear periphery helps mediate P-cell nuclear migration though constricted spaces in Caenorhabditis elegans.

Nuclei adjust their deformability while migrating through constrictions to enable structural changes and maintain nuclear integrity. The effect of heterochromatin anchored at the nucleoplasmic face of the inner nuclear membrane on nuclear morphology and deformability during in vivo nuclear migration through constricted spaces remains unclear. Here, we show that abolishing peripheral heterochromatin anchorage by eliminating CEC-4, a chromodomain protein that tethers H3K9-methylated chromatin to the nuclear periphery, disrupts constrained P-cell nuclear migration in Caenorhabditis elegans larvae in the absence of the established LINC complex-dependent pathway. CEC-4 acts in parallel to an actin and CDC-42-based pathway. We also demonstrate the necessity for the chromatin methyltransferases MET-2 and JMJD-1.2 during P-cell nuclear migration in the absence of functional LINC complexes. We conclude that H3K9-nethylated chromatin needs to be anchored to the nucleoplasmic face of the inner nuclear membrane to help facilitate nuclear migration through constricted spaces in vivo.

Exploring the antifungal, antibiofilm and antienzymatic potential of Rottlerin in an in vitro and in vivo approach

Candida species have been responsible for a high number of invasive infections worldwide. In this sense, Rottlerin has demonstrated a wide range of pharmacological activities. Therefore, this study aimed to evaluate the antifungal, antibiofilm and antivirulence activity of Rottlerin in vitro against Candida spp. and its toxicity and antifungal activity in vivo. Rottlerin showed antifungal activity against all yeasts evaluated, presenting Minimum Inhibitory and Fungicidal Concentration (MIC and MFC) values of 7.81 to > 1000 µg/mL. Futhermore, it was able to significantly inhibit biofilm production, presenting Biofilm Inhibitory Concentration (MICB50) values that ranged from 15.62 to 250 µg/mL and inhibition of the cell viability of the biofilm by 50% (IC50) from 2.24 to 12.76 µg/mL. There was a considerable reduction in all hydrolytic enzymes evaluated, with emphasis on hemolysin where Rottlerin showed a reduction of up to 20%. In the scanning electron microscopy (SEM) analysis, Rottlerin was able to completely inhibit filamentation by C. albicans. Regarding in vivo tests, Rottlerin did not demonstrate toxicity at the therapeutic concentrations demonstrated here and was able to increase the survival of C. elegans larvae infected. The results herein presented are innovative and pioneering in terms of Rottlerin’s multipotentiality against these fungal infections.

Label-free Brillouin endo-microscopy for the quantitative 3D imaging of sub-micrometre biology

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 μm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.

Magnetic Control of Nonmagnetic Living Organisms.

Living organisms inspire the design of microrobots, but their functionality is unmatched. Next-generation microrobots aim to leverage the sensing and communication abilities of organisms through magnetic hybridization, attaching magnetic particles to them for external control. However, the protocols used for magnetic hybridization are morphology specific and are not generalizable. We propose an alternative approach that leverages the principles of negative magnetostatics and magnetophoresis to control nonmagnetic organisms with external magnetic fields. To do this, we disperse model organisms in dispersions of Fe3O4 nanoparticles and expose them to either uniform or gradient magnetic fields. In uniform magnetic fields, living organisms align with the field due to external torque, while gradient magnetic fields generate a negative magnetophoretic force, pushing objects away from external magnets. The magnetic fields enable controlling the position and orientation of Caenorhabditis elegans larvae and flagellated bacteria through directional interactions and magnitude. This control is diminished in live spermatozoa and adult C. elegans due to stronger internal biological activity, i.e., force/torque. Our study presents a method for spatiotemporal organization of living organisms without requiring magnetic hybridization, opening the way for the development of controllable living microbiorobots.

Ets-10p::gfp expression is predictive of dauer formation in daf-16; daf-7 larvae.

In adverse conditions, Caenorhabditis elegans larvae can enter the alternative L2d stage. If conditions remain poor, L2d larvae can molt into stress-resistant dauer larvae. The FOXO ortholog daf-16 promotes dauer formation, but daf-16 mutants can enter dauer with incomplete penetrance in combination with a mutation in daf-7 /TGFβ. The degree to which daf-16 ; daf-7 larvae enter L2d is unknown. Here we show that many daf-16 ; daf-7 mutants express intermediate levels of the ets-10p::gfp L2d marker, suggesting incomplete entry into L2d. Furthermore, lack of ets-10p::gfp expression early in the second larval stage partially predicts which daf-16 ; daf-7 larvae will bypass dauer formation.

Simira cordifolia protects against metal induced-toxicity in Caenorhabditis elegans.

Simira cordifolia (Hook.f.) Steyerm (Rubiaceae) is a vascular plant used in Northern Colombia as a source of pigments and wood. However, there is a lack of information regarding its pharmacology and toxicity. This research aimed to study the hydroalcoholic extract of Simira cordifolia as a protector against metal-induced toxicity in Caenorhabditis elegans. Preliminary phytochemical screening of the hydroalcoholic extract of S. cordifolia (HAE-Sc) was conducted using HPLC-ESI-QTOF. Wild-type N2 C. elegans larvae were exposed to different concentrations of HAE-Sc evaluating lethality (50-5000μg/mL), growth, lifespan, resistance to heat stress, and its protective effect against Mercury (Hg)-, Lead (Pb)- and Cadmium (Cd)-induced lethality (50-1000μg/mL). The main metabolites present in the extract were iridoids, β-carboline-alkaloids and polyphenols. Bioassays demonstrated that HAE-Sc exhibited low toxicity, with significant lethality (4.2% and 9.4%) occurring at 2500-5000μg/mL. Growth inhibition reached up to 23.3%, while reproduction declined 13% and 17% at concentrations 500 and 1000μg/mL, respectively. HAE-Sc enhanced the survival rate of the nematode under thermal stress by up to 79.8%, and extended the mean lifespan of worms by over 33% compared to control. The average lifespan was prolonged by 15.3% and 18.5% at 50 and 100μg/mL HAE-Sc, respectively. The extract (1000μg/mL) was able to reduce the death of C. elegans in the presence of heavy metals up to 65.9, 96.8% and 87% for Pb, Hg, and Cd, respectively. In summary, S. cordifolia shows potential protective effects in C. elegans against toxicity caused by heavy metals and heat.

The epithelial Na+ channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components to new boutons in remodeling neurons

Circuit refinement involves the formation of new presynaptic boutons as others are dismantled. Nascent presynaptic sites can incorporate material from recently eliminated synapses, but the recycling mechanisms remain elusive. In early-stage C. elegans larvae, the presynaptic boutons of GABAergic DD neurons are removed and new outputs established at alternative sites. Here, we show that developmentally regulated expression of the epithelial Na+ channel (ENaC) UNC-8 in remodeling DD neurons promotes a Ca2+ and actin-dependent mechanism, involving activity-dependent bulk endocytosis (ADBE), that recycles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. In contrast, we find that an ADBE-like mechanism results in the distal recycling of synaptic material from old to new synapses. Thus, our findings suggest that a native mechanism (ADBE) can be repurposed to dismantle presynaptic terminals for reassembly at new, distant locations.

Antioxidant, angiotensin converting enzyme inhibitory and anthelmintic activities of bark essential oil of Cinnamomum bejolghota (Buch.-Ham.) Sweet from North East India

Cinnamomum bejolghota (Buch.-Ham.) Sweetisa traditional anthelmintic and cardiotonic medicinal plant. The essential oil extracted from the bark of C. bejolghota was characterized by gas chromatography-mass spectrometry and 28 constituents were identified. Monoterpenoids were found to be dominating chemical constituents with α-terpineol (18.57 %) as the major component of the essential oil. The antioxidant activity of the essential oil was evaluated by DPPH radical, β-carotene bleaching and reducing power assay methods. The essential oil showed in-vitro dose-response hindering activity against angiotensin-converting enzyme with an IC50 value of 33.43 ± 0.46 µg/mL.The essential oil also exhibited potent anthelmintic property in-vitro against Caenorhabditis elegans with an ED50value of 80.53 ± 3.57 µg/mLin adult mortality assay. The inhibitory activity of essential oil against larval development assay showed an ED50value of 72.40 ± 2.68 µg/mL against C. elegans larva. Further, the egg hatching assay using C. eleganseggs demonstrated an ED50 value of 68.27 ± 3.51 µg/mL with this essential oil. Thus, the ethnomedicinal claim has been validated for the bark essential oil, which provides an excellent scope for the development of the C. bejolghota bark essential oil as novel herbal antihypertensive and anthelmintic agents.

A bacterial pathogen induces developmental slowing by high reactive oxygen species and mitochondrial dysfunction in Caenorhabditis elegans

Host-pathogen interactions are complex by nature, and the host developmental stage increases this complexity. By utilizing Caenorhabditis elegans larvae as the host and the bacterium Pseudomonas aeruginosa as the pathogen, we investigated how a developing organism copes with pathogenic stress. By screening 36 P.aeruginosa isolates, we found that the CF18 strain causes a severe but reversible developmental delay via induction of reactive oxygen species (ROS) and mitochondrial dysfunction. While the larvae upregulate mitophagy, antimicrobial, and detoxification genes, mitochondrial unfolded protein response (UPRmt) genes are repressed. Either antioxidant or iron supplementation rescues the phenotypes. We examined the virulence factors of CF18 via transposon mutagenesis and RNA sequencing (RNA-seq). We found that non-phenazine toxins that are regulated by quorum sensing (QS) and the GacA/S system are responsible for developmental slowing. This study highlights the importance of ROS levels and mitochondrial health as determinants of developmental rate and how pathogens can attack these important features.

Chemical cannibalistic cues make damselfly larvae hide rather than hunt

Adopting cannibalism substantially affects individual fitness, and recognizing the presence of other cannibals provides additional benefits such as the opportunity to prepare for hunting or defense. This recognition can be facilitated by perceiving conspecific chemical cues. Their role in cannibalistic interactions is less studied than in interspecific predation and it is unclear whether these cues inform individuals of danger or of food availability. Interpretation of these cues is crucial to balance the costs and benefits of anti-predator and feeding strategies, which can directly influence individual fitness. In this study we aimed to test whether damselfly larvae shift towards bolder and more exploratory (cannibalistic) behavior, or become more careful to avoid potential cannibals (as prey) in response to such cues. We conducted behavioral and respiratory experiments with Ischnura elegans larvae to investigate their response to chemical cues from older and larger conspecific larvae. We found that I. elegans larvae decrease their activity and shift their respiratory-related behavior, indicating activation of anti-predator defense mechanisms in response to conspecific chemical cues. Our findings indicate that individuals exposed to conspecific chemical cues balance catching prey with staying safe.

Nematicidal lipopeptides from Bacillus paralicheniformis and Bacillus subtilis: A comparative study.

The aim of this work was to develop a comparative study between Bacillus paralicheniformis TB197 and B. subtilis ATCC 21332 strains in terms of growth, cyclic lipopeptide production, nematicidal activity, and active lipopeptide characteristics. Crude lipopeptide extracts (CLEs) from their fermentation broths were obtained, and their nematicidal activity (NA) was estimated as the mean lethal dose (LD50), employing Caenorhabditis elegans. Using a bioguided approach, CLE components were fractionated by semipreparative thin layer chromatography, and active lipopeptides were characterized by mass spectrometry. Both strains produced similar concentrations of CLEs (p ≥ 0.05) (0.99 ± 0.11 and 1.14 ± 0.15mg/mL by TB197 and ATCC 21332, respectively). The estimated LD50 values of CLEs from the TB197 and ATCC 21332 strains were 3.88 and 8.15mg/mL, respectively, showing that the NA of the TB197 strain CLE was 2.1-fold higher (p ≤ 0.05). Mass spectrometry revealed that strain TB197 synthesizes several families of lipopeptides, namely, fengycin A (C14-C17), fengycin B (C16-C17), surfactin (C15-C17), and lichenysin (C12, C13, C14, and C16), from which fengycins and lichenysins possess the highest NA (100 and 60% mortality in C. elegans larvae, respectively), while the ATCC 21332 strain produces mainly surfactin (C13-C17) (NA 63% mortality). The main differences found in this study were that the TB197 strain has a higher tolerance to inhibition by the product, and the lipopeptides they synthesize have a higher nematicidal activity due to the diversity of families compared to ATCC 21332. Likewise, it was shown that more polar lipopeptides (fengycins) are more effective at causing mortality in C. elegans larvae. KEY POINTS: • The nematicidal activity of lipopeptides from TB197 is higher than from ATCC 21332 • TB197 produces surfactin, lichenysin, and fengycin, while ATCC 21332 mainly produces surfactin • The most polar lipopeptides (fengycins) cause more mortality in C. elegans L2.

EVALUATION OF THE GEROPROTECTIVE EFFECT OF SQUALENE ON THE CAENORHABDITIS ELEGANS MODEL

Background. Increasing healthy life expectancy is a priority in modern healthcare. Squalene, a triterpene exhibiting antioxidant, anti-inflammatory and other properties, is a promising candidate for geroprotectors.
 Purpose. Assess the activity of squalene, that is, the possibility of its further use as a geroprotector.
 Materials and methods. Squalene was purchased from Sigma-Aldrich (USA) for research. Squalene solutions were prepared with concentrations of 10, 50, 100, 200, 400, 800, and 1600 µmol/L (solvent dimethyl sulfoxide—DMSO). The work was carried out on the basis of the Laboratory for Biotesting of Natural Nutraceuticals (KemGU, Russia). Antioxidant activity was assessed by capturing ABTS radical cations in comparison with the activity of ascorbic acid. The model organism Caenorhabditis elegans N2 Bristol was purchased from the Laboratory for the Development of Innovative Medicines and Agrobiotechnologies of the Moscow Institute of Physics and Technology. To assess the stress resistance of nematodes during cultivation, conditions of thermal (33°C) and oxidative (adding 15 μl of 1 M paraquat, 20°C) stress were created for 24 and 48 h. conditions (20 °C) for 61 days. The statistical significance of the results was assessed using Student’s t-test (in Microsoft Office Excel 2007). Lifespan data were assessed using a Kaplan-Meier survival plot combined with a log-rank test (Mantel-Cox). Data processing was carried out using the OASIS online application (https://sbi.postech.ac.kr/oasis/).
 Results. The results showed that squalene solutions did not exhibit antioxidant properties; did not trap radical cations with ABTS. A solution of squalene at a concentration of 10 μM contributed to an increase in the stress resistance of nematodes. Under conditions of oxidative stress, the survival of nematodes during cultivation for 24 hours is 3.1% higher than in the control. Under conditions of heat stress during cultivation for 48 hours, the survival rate is higher by 11.3%. Squalene solutions did not affect the lifespan of nematodes.
 Conclusion. Presumably, the antioxidant activity of squalene is expressed not in the capture of free radicals, but in the ability to act on biomolecules that lead to oxidative stress. A squalene solution with a concentration of 10 μM can reduce the negative impact of one of the factors causing premature aging. In order to assess the possibility of using squalene as a geroprotector without relying on cation-radical capture, further studies are needed to study the impact of the substance on other targets, mechanisms of action related to the occurrence of age-associated diseases. For example, analysis of the effect of squalene solutions on the growth rate of C. elegans larvae, the ability to influence the expression of antioxidant defense genes, the presence of anti-inflammatory and other properties.

Long-term cryostorage does not negatively affect the recovery of Caenorhabditis elegans

Cryostorage of Caenorhabditis elegans nematodes is important to maintain the many lines used for research. The standard method uses 15% of glycerol in M9-Buffer and a cooling rate of 1 °C/min; then worms can be stored in a −80 °C freezer or in liquid nitrogen. The recovery of C. elegans from stocks stored in liquid nitrogen is reported to be in the range of 35–45% and slightly decreases after years of storage. The storage at −80 °C is also considered safe, but the recovery is not as high as in liquid nitrogen. These observations have not been experimentally reported and therefore require verification. In this study, the standard methods were used in a set of experiments to compare the recovery of larvae and adult worms stored at −80 °C or in liquid nitrogen, after short- (a week) or long-term storage (3.5 years). No differences were observed in recovery, either for the time of storage or for the temperature of storage. Recovery of larvae was 32% at −80 °C and 36% in liquid nitrogen after 3.5 yr and that was not significantly different from the 7-d recovery rates. Adult worm recovery was below 5% for all treatments. These results suggest that both methods of storage can be used to successfully store C. elegans larvae for at least 3.5 years.

EGFR signal transduction is downregulated in C. elegans vulval precursor cells during dauer diapause.

Caenorhabditis elegans larvae display developmental plasticity in response to environmental conditions: in adverse conditions, second-stage larvae enter a reversible, long-lived dauer stage instead of proceeding to reproductive adulthood. Dauer entry interrupts vulval induction and is associated with a reprogramming-like event that preserves the multipotency of vulval precursor cells (VPCs), allowing vulval development to reinitiate if conditions improve. Vulval induction requires the LIN-3/EGF-like signal from the gonad, which activates EGFR-Ras-ERK signal transduction in the nearest VPC, P6.p. Here, using a biosensor and live imaging we show that EGFR-Ras-ERK activity is downregulated in P6.p in dauers. We investigated this process using gene mutations or transgenes to manipulate different steps of the pathway, and by analyzing LET-23/EGFR subcellular localization during dauer life history. We found that the response to EGF is attenuated at or upstream of Ras activation, and discuss potential membrane-associated mechanisms that could achieve this. We also describe other findings pertaining to the maintenance of VPC competence and quiescence in dauer larvae. Our analysis indicates that VPCs have L2-like and unique dauer stage features rather than features of L3 VPCs in continuous development.

Antifungal Activities of Essential Oils from Protium ovatum Engl. Against Malassezia furfur and Candida Species

Fungal opportunistic infections have increased in recent decades due to the increase in the immunosuppressed patients and the indiscriminate use of antifungals. In Brazil, a country with the greatest biodiversity in the world, studies that seek new antifungals from natural sources have been stimulated. Protium ovatum Engl., belongs to the Burseraceae family and is a shrub tree found mainly in Brazil, in the Cerrado biome, and has medicinal, food and aromatic uses. This study aims to investigate the chemical composition and the anti-Malassezia furfur and anticandidal activities of essential oils (EOs) from Protium ovatum ripe fruit (RF-EO) and unripe fruit (UF-EO). The EOs antifungal activities were determined by microdilution broth methodology. GC-FID and GC-MS analyses showed that limonene, α-pinene and myrcene were the major components of both EOs. MIC values of RF-EO and UF-EO against M. furfur were 375 and 1500 µg/mL, respectively. RF-EO exhibited MIC values between 62.5 and 250 µg/mL while UF-EO was slightly active (> 1000 µg/mL) against Candida species. In addition, RF-EO showed antibiofilm activity against Candida species and was not toxic to C. elegans larvae. This study suggests that EOs from P. ovatum could be an alternative therapeutic option for infectious diseases caused by M. furfur and Candida species.

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults.

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.