4.1. Immobilization and Imaging
C. elegans are transparent animals, which allows any researcher to perform in vivo visualization of a fluorescently tagged protein that is transgenically expressed and serves as a greater, yet simple, in vivo model organism [
16]. Such live observations facilitate detailed understanding of several subcellular functions and molecular interactions of the fluorescently tagged protein. In order to achieve successful imaging of living worms, it is absolutely necessary to immobilize the worms. A live worm swims constantly in the buffer solution in which it is suspended. However, the need of immobilizing the worm also depends on experimental needs—for example, neuronal activity imaging in certain cases requires a non-immobilized worm, like calcium imaging so as to understand neuronal activity and behavior [
34]. Methods and software have been developed to facilitate the imaging of mobile worms [
34]. A simple, cost-effective method of immobilizing a worm for a short time, so as to achieve in vivo imaging, is followed by all
C. elegans-based research laboratories. According to this method, researchers typically sandwich the worms between a 2% agarose-coated glass slide and a cover-glass, along with an appropriate buffer or M9 buffer (3 g KH
2PO
4, 6 g Na
2HPO
4, 5 g NaCl, 1 mL 1 M MgSO
4, H
2O to 1 L, sterilize by autoclaving) containing an anesthetic, such as levamisole, tetramisole, etc. [
35]. Unfortunately, using this method for live in vivo imaging is not possible over a longer period, as the worms’ vital signs deteriorate and the anesthesia is shown to affect the cellular physiology [
20]. Further, researchers cannot use this method for screening purposes, as it is almost impossible to recover a particular worm from the sandwich. In order to overcome these disadvantages of the above mentioned procedure, several types of methods have been developed, whereby researchers can perform live worm imaging for longer time periods without anesthesia, or screen the worm for a specific phenotype and recover it for further analysis. However, it was difficult to overcome all the disadvantages simultaneously until microfluidic devices for long-term immobilization and imaging were invented and developed. All new procedures following the use of microfluidic devices reduce the damage caused to the worms, though not completely. Further new microfluidic devices always aim at increasing performance and thereby providing quicker recovery and survival rates for the worms.
Chokshi et al [
36]. developed a simple worm chip system to efficiently immobilize the worms in addition to studying the motility behavior of
C. elegans. This system involves two layers of PDMS on a glass slide with the appropriate chambers and channels for fluid flow (
Figure 1). The worm is immobilized via two different techniques: the first utilizes the diffusion property of PDMS to diffuse CO
2 from the upper PDMS channel to the lower channel containing the worm, thereby immobilizing the worm; the second employs the collapsible nature of PDMS to mechanically compress the worm by increasing the air pressure on the upper PDMS fluidic channel [
36]. Following immobilization of the worm of up to an hour, Chokshi et al. [
36] quantified the damage caused by the immobilization by evaluating the locomotive speed of the worm. They found that immobilization using CO
2 caused less damage to the worm than the application of mechanical pressure. The average speed of the worm after immobilization for an hour was reduced to 0% and 70%, respectively, upon remobilization using the collapsible PDMS and CO
2 methods. Mechanical immobilization of worms using the collapsible property of PDMS and then successful imaging of intracellular transport was performed, demonstrating the easy handling and versatility of microfluidic worm chips [
9,
37].
Although microfluidic systems using valves, channels and chambers are very efficient for the handling of adult worms, studies involving the larval stage of worms are challenging, given that they are much smaller than adult worms. Very small first larval stage (L1) worms can easily clog the microfluidic channels and have less efficient trapping. In order to overcome the challenge of larval stage worms, Aubry et al. combined the temperature-sensitive reversible gelation of Pluronic F27 with microfluidic channels [
38]. Initially, this chip works by trapping a single worm in the production unit with Pluronic F27 (
Figure 2). As the worm enters the storage unit of the chip, one can adjust the temperature to solidify the gel, resulting in immobilization of the worm and thereby allowing for efficient imaging. Finally, researchers can sort the worm in the sorting unit of the chip. We present a schematic representation of a working model of this device in
Figure 3.
Cornaglia et al. introduced a device for immobilization and imaging of
C. elegans embryos to study their development and the molecular changes that take place during embryogenesis. This device consists of a worm culture chamber, where one can culture adult worms, and an array of micro-compartments called embryo-chambers, where researchers immobilize a single embryo and examine it in real time throughout its development. An array of embryo-chambers in this device also allows for the performance of high throughput (HTP) analysis by permitting researchers to analyze several embryos at the same time [
39].
Separate to the above-mentioned microfluidic devices, researchers have attempted to design HTP microfluidic platforms for the immobilization and imaging of several worms simultaneously, leading to some successful device development. For example, the microfluidic devices described by Hulme et al. [
40] (
Figure 4) and Lee et al. [
41] (
Figure 5 and
Figure 6), use several arrays of narrow microchannels to entrap
C. elegans, resulting in immobilization. Hulme et al. also monitored the survival of the worm after immobilization in their device, as they expected that the pressure exerted on the worm could possibly damage its cuticle and internal structures. By quantifying the number of days of survival of the worm and the number of offspring it created, Hulme et al. demonstrated that the worms exhibited normal function following immobilization. However, analysis at the gene expression level for stress response could provide a better understanding of the damage potentially caused by microfluidic devices to worms.
4.3. Behavior Analysis
Researchers have developed numerous traditional behavioral assays for
C. elegans since the introduction of
C. elegans as a model organism.
C. elegans have 302 neurons and the behavior of
C. elegans in response to a particular stimuli reflects its neuronal and muscular functions and health [
44]. Some common behaviors these researchers analyze include mechanosensation, osmotic avoidance, chemotaxis, electrotaxis, feeding response, thermal response, egg-laying, mating and reproductive behavior, learning and memory, defecation, etc. [
44]. Following the development of microfluidics chips, we can obtain certain levels of accuracy in several of the behavioral assays—for example, dissection of precise concentrations of chemoattractant. Recent technological developments have provided advanced microfluidic chips that can help elucidate worm behaviors such as electrotaxis [
45,
46], chemotaxis [
47,
48], stress response behavior during crowding [
49,
50], neuronal pathway and subsequent behavioral responses [
51], host invasion behavior [
19], etc.
Although examination of chemotaxis behavior is a very common traditional assay (for example, quadrant assay [
52]), the calculation of the accurate concentration or development of proper concentration gradient of chemical stimuli employed is, in most cases, difficult to determine. Wang et al. developed a microfluidic/nanofluidic device in which the nanochannels can develop a concentration gradient to test the chemical [
47].
Wang et al. developed an array of micro-columns-based microfluidic system to study a worm’s response to a crowded environment (
Figure 8) [
50]. The array of columns in the microfluidic chip provides sufficient mechanosensation to the worm to prompt a crowding stress response. The distance between the array of micro-columns can be adjusted, to create an appropriately crowded environment so as to induce a stress response at the cellular level, such as translocation of the fluorescently tagged protein, DAF-16::GFP, into the nuclear subcellular location, which researchers can then analyze by imaging the live worm at different time points. Crowding induces stress, in turn, affects the physiological behavior.
Another interesting device developed by Chuang et al. exploits the electrotaxis behavior of worms to induce a physically active environment, producing a “worm treadmill” effect. The physical activity produced in this manner is equivalent to exercise and research shows that this effect can protect the worm from age-related cellular degeneration. In this worm chip, where the worms are subjected to an electric field, the worms are attracted towards the cathode via electrotaxis in the microchannels. The polarity of the electric field can be switched between the electrodes so that the worms are attracted to the new cathode in turn, thereby producing the treadmill effect [
21].
The combination of microfluidic devices and optogenetics had taken our understanding of the functional relationship between proteins and worm behavior to a whole new level. Several
C. elegans mutant worms exhibit uncoordinated behavior and researchers have mapped the gene responsible for such behavior. However, while Hwang et al. examined the clear relationship between several muscle proteins and their collaborative role in behavior through the analysis of muscle kinetics, contraction and relaxation by subjecting an ontogenetically manipulable worm in microfluidic channels, other methods of obtaining such data do not exist [
53].
4.4. Drug Screening and Toxicological Studies
C. elegans is a model organism with a genome ~65% similar to human disease genes [
54,
55]. For this reason, researchers use it for drug screening and toxicity examination of various chemical compounds. Varieties of worm chips are developed for real-time drug identification, screening and for testing toxicity based on worm behavior [
56,
57,
58,
59], electrophysiological signals [
60], aging indications [
61], antimicrobial or metabolic activity [
62] and physical toxicity [
63]. Thus, researchers use microfluidic systems not only on
C. elegans, but also on other worm parasites for therapeutic agent development [
56,
58].
Yang et al. developed a worm chip for screening and evaluating in vivo antimicrobial activity. The authors fabricated two layers of radial worm chips, radiating from a central reservoir to 32 chambers, which in turn were connected to drug delivery inlets from four concentration gradient generators (CGG). This worm chip can simultaneously screen 32 concentration gradients with four types of drugs (
Figure 9). One can load the worms from the central reservoir, culture them in the chamber and analyze them simultaneously [
62].
4.5. Microsurgery
Understanding the factors that play vital roles in neuronal regeneration has gained importance in the search of cures for several forms of neurodegenerative diseases and neuronal injuries. Researchers have developed
C. elegans as one of the model organisms for studying the neuronal regeneration process, using femtosecond laser pulses to create precise ablation of the neurons [
64]. Researchers are constantly developing and automating various microfluidic devices for worm handling so as to avoid the use of any anesthesia for neuronal axotomy [
65,
66]. One can also use some of the abovementioned worm chips for the immobilization of worms to immobilize worms for laser axotomy (refer to the “immobilization and imaging” section, Lee et al. [
41]). Here, we can briefly outline a new automated microfluidic chip for laser microsurgery in worms. Gokce et al. designed a two-layer microfluidic system, in which the bottom layer, called the flow layer, transports the worm to different areas of the devices for immobilization, laser axotomy and removal, etc. The top layer is called the control layer and its primary function is to immobilize the worm through controlled valves upon pressurization. One can mount the chip on the microscope stage to perform laser severing using a femtosecond laser with a wavelength of 802 nm and 1 KHz pulsing frequency. The system connects to a pressurized external fluid chamber in order to control the flow rate into the microfluidic chip. The system is fully automated and one can control it via custom-written code, run by LabVIEW (National Instruments, Austin, TX, USA) [
66].
4.6. Worm/C. elegans Sorting
Well-established genetic analysis has proven
C. elegans to be a powerful genetic tool. Screening of new genes using both forward and reverse genetic screening is now a popular technique for understanding molecular pathways and protein functions in several genetic laboratories. Screening for a particular phenotype among several hundreds or thousands of worms is very laborious and time-consuming, but microfluidic systems have the power to greatly reduce the screening time and enable quick recovery/sorting of the observed genetic variant. The operation of worm sorting chips is based on various characteristic phenotypes of the worms, such as the visible microscopic phenotype of genetic variants, or the electrotaxis, behavioral phenotype, size and motility behavior of the worm, etc. Besides sorting worms after genetic screening [
67,
68], researchers use worm sorting chips to sort worms based on age [
69], size [
70,
71], sex, motility [
72], electrophysiological characteristics [
73,
74], etc. Researchers can make use of these different types of sorting in various metabolic, molecular biology and population studies.
Aubry et al. described a device (see the “Immobilization and Imaging” section) that can also be used for sorting worms that possess microscopic visible phenotypic variations after screening (
Figure 2 and
Figure 3) [
38]. Aubry et al. characterized this method of sorting as “active” sorting of worms, whereby a researcher or computerized system actively identifies the worm, based on a fluorescent signal, that they must separate from the population of worms.
Rezai et al. described a single-layer microfluidic chip for worm sorting based on the electrotaxis behavior of the worms (
Figure 10). This device can efficiently sort wild type worms at different developmental stages and screen for genetic mutant variants, which display defective neuronal and muscular activity as compared to wild type populations. Thus, the device can age synchronize the worms while screening for genetic mutation with defective neuronal and muscle activity [
73]. The authors describe a passive method for sorting the worms. Unlike active sorting, the Rezai et al [
73] device involves the separation of worms solely based on the differential attraction of worms towards an electrical signal, rendering this approach a passive sorting method. Yet another device designed by Casadevall I Solva et al [
69]. works passively for age- and size-based synchronization of worms, and can achieve a rate of 200–1200 worms per minute with differential efficiency [
69]. This device consists of pillar arrays, pools, smart filters and mazes in the flow chamber. As the worms pass through these obstacles they are segregated such that, for example, only larval worms are sorted into one particular channel, while adults are sorted into another.
Although there are different types of microfluidic chips available for size-dependent sorting of worm populations, each device varies in its structural design, design complexity, efficient worm handling and rate of sorting. Dong et al. designed a two-layer microfluidic chip in which an external pressure-based deformation property on a PDMS layer sorts the worms, based on the size of the space between the two chambers (
Figure 11) [
71]. Changes in the pressure of the control layer cause appropriate changes in the size of the channel in the PDMS flow layer, thereby allowing only those worms of a particular size to pass between the channel-separated chambers. It is necessary to standardize the precise optimization of pressure for particular worm size. The size-dependent sorting efficiency of this chip is 3.5 worms per second.