How does the ScreenChip System work?
The pharynx is a heart-like neuromuscular organ involved in nematodes’ feeding behavior. During a pharyngeal muscle contraction, the ion exchange underlying the pharyngeal action potential generates a change of voltage in the worm’s direct environment. The ScreenChip System detects such voltage change using external electrodes placed onto the glass floor of the microfluidic chips (Lockery et el., 2012) and directs it to the acquisition software. The design of the chips ensures a snug fit of the worm to create a sufficiently large electrical resistance to record the electrical activity with a high signal-to-noise ratio.
You will need to connect the outlet of your ScreenChip System to a vacuum source (portable vacuum pump or house vacuum tap). The worms are moved from the epitube and through the chips by the negative pressure generated by the vacuum source. Each worm is carried by the flowing fluid until it reaches the constriction part of the main channel (“trap”). Worms are then mechanically held in position throughout an experiment.
Using the ScreenChip system does not require intensive training and users usually get usable data on the first day of use. We stay in touch with our customers to help you with your experimental design or with the platform set-up if needed.
In order to record feeding behavior, pharyngeal activity must be induced. This can be achieved by including either the neuromodulator serotonin or fresh bacteria in the saline medium. Inducing pharyngeal pumping allows you to study the effect of various environmental components such as bacterial strains and drugs. Simply incubate or cultivate your worms into the desired environmental conditions and load the worm.
Each worm can be recorded, one at a time, for several minutes up to 24 hours. At the end of each recording, vacuum each worm out of the chip while loading the next worm by using the vacuum source.
On average, we have observed that it takes 30-60 seconds to load 50 worms into the first loading chamber within the chip. Once all the worms are loaded in the chamber, it takes on average less than 10 seconds to move a worm into the recording channel.
The ScreenChip System is composed of two key elements: a custom dock with electrical components and a mini amplifier. When you purchase the ScreenChip starter kit, you will receive all of the elements that you will need to start your experiments right away. Just install our free data acquisition software to start recording your data.
We designed the ScreenChip System to fit entirely on the stage of a dissection microscope.
Please note that in order to load worms within the ScreenChip, you will need a vacuum source, which is not included in the starter kit as many labs already own one. However, we are happy to provide you with a vacuum pump if needed, click here for more information.
The ScreenChips are composed of polydimethylsiloxane (PDMS), a completely optically clear and gas permeable material, bonded on a glass slide. Another distinct characteristic of PDMS is its ability to absorb hydrophobic small molecules. Re-using chips beyond the recommended time of 8 hours could result in an overall change in solution concentrations and could potentially alter experimental outcomes.
How to acquire and analyze data
As in the vertebrate heart, the pattern of rhythmic contractions of the nematode pharynx is primarily generated by the pharyngeal muscles themselves. Each contraction is associated with an action potential, a large voltage transient that can be recorded by electrodes placed on the surface of the body. By analogy to an electrocardiogram, such a recording in nematodes is called an electropharyngeogram (EPG)(Lockery et al., 2012). In addition to registering action potentials in the pharyngeal muscles, the EPG also registers the activity of neurons that regulate the rate of pharyngeal pumping in much the same way as the autonomic nervous system regulates the vertebrate heart. Thus, the EPG can be used to investigate the effects of drugs that act on neurons as well as muscles.
The ScreenChips are completely optically clear and allow you to perform imaging experiments up to 20X with an inverted microscope or with a dissecting microscope.
We do offer an imaging solution with our imaging chip, which allows you to take high resolution images of your worms using brightfield or fluorescence. The imaging chip does not contain electrodes, and therefore does not let you records EPGs.
On average, ScreenChip user record 50-70 worms with one chip. We recommend changing the chips after about 8 hours of use.
The data acquisition process is rapid and simple as presented here. Both data acquisition and analysis are automated through our free, open source softwares: NemAcquire and NemAnalaysis. NemAcquire allows you to monitor each worm’s feeding behavior in real time, while recording 500 data points per second. Data from each worm is recorded in a .txt file that can be read by multiple free softwares, including NemAnalysis.
NemAnalysis is designed to help you get the most information out of your data, while allowing you to control how your data is analyzed. NemAnalysis automatically extracts the data from NemAcquire files to identify and measure pharyngeal pumping events and quantify various aspects of the feeding pattern of your worms (changes of pumping frequency over time, consistency of feeding, duration of pharyngeal contractions etc…).
NemAnalysis lets you extract raw and analyzed data from your worms into an Excel sheet, so you can perform the statistical analyses of your choice.
Using the ScreenChip System, you will be able to extract:
- The pumping pattern of your worms
- The interpump interval duration (pauses)
- Pump duration
- Pumping frequency (rate)
- Frequency changes throughout your recording
You will also be able to export your acquired data into an Excel sheet
What worms are compatible with the ScreenChip System?
We have recorded feeding activity for multiple nematode parasitic and free-living species such as C. elegans, and various species of hookworms larvae, Panagrellus redivivus or Pristionchus pacificus.
Is the wMicroTracker compatible with all worm strains?
The system is compatible with most worm strains including wild nematode isolates and parasitic nematodes. Non motile/non-good swimming strains (such as roll or severe unc mutants) are not recommended.
Can I perform worm development assays with a wMicroTracker?
Yes, the wMicroTracker can measure worm activity form L1 to older adult for weeks. Be sure to validate your liquid culture protocol before using it in the WMicroTracker.
Can I measure the worm’s curvature/body bends with the wMicroTracker?
Although the wMicroTracker does not have a camera, it is possible to correlate the wMicroTracker’s data (number of beam breaks) to the number of body bends per minute.
Is it possible to measure individual worms?
It is not recommended. However, it is possible to perform experiments with as few as 1 worm. See graphs in this Tech Note for more details.
Can I use bacteria in the well and still get accurate measurements of movement?
Yes. Worms can be measured in the wMicroTracker for multiple days when placed in bacteria. We recommend that you use fresh bacteria at an OD600 of 0.5 (up to 1). Under these conditions, adult worms usually have enough food for 3-5 days. Another option is to cultivate your worms in axenic media (CeMM) instead of using bacteria.
How long can I keep and measure my worms in the wMicroTracker?
The wMicroTracker can acquire data on your worms for weeks without interruption. The limiting factor for longitudinal studies will be determined by your worm culture protocol and requirements. For long term data acquisition in the wMicroTracker, we recommend that you place your worms in axenic media. We recommend that you conduct measurements for at least 100 minutes to reduce standard error. See protocol for healthspan assays for more information.
Do the beams maintain the same properties over time?
We observed no degradation of the LEDs over time. The LEDs in the wMicroTrackers have a lifetime of 36,000 hours. Since they flash every 1/384 sec, this is equivalent of 10 years of use. The wMicroTracker was engineered to auto-compensate and auto-calibrate the beams to maintain signal linearity over time.
Do worms develop an aversion to the beam over time?
The infra-red beams in the wMicroTracker are generated by low-power LEDs and have been shown to be non-invasive for C. elegans (Simonetta and Golombek 2007).
Is the software free?
Yes, you can download the wMicroTracker software here.
What data do I get from the wMicroTracker?
The LED beams pulse every 1/384 seconds and the software records every interruptions of the beams. This data is pooled and analyzed when the data report is generated by the software and presented as “average activity count by data interval”. You can always change the analysis bin (data interval) size after the data is collected. The minimum bin size recommended for analysis is 5 minutes. Choosing smaller bins may increase variability of your data.
What is the beam size relative to the worms?
The LED beams have a diameter of 150µm, which is larger than the diameter of an adult C. elegans worm (100µm on average).
What are the beam specs?
Wavelength: Infrared, 880nm
- Temperature: no heat generated
- Pulse frequency: 1/384 sec
- Number of beams per well for the wMicroTracker:
- 96-well plate: 2 beams per well
- 384-well plate: 1 beam per well
These properties cannot be modified.
What are the computer requirements to use the wMicroTracker?
Computer minimum requirements:
- Pentium II processor or above (>1GHz clock)
- 512Mb of RAM memory
- 1 USB port
- Windows XP 32 bits (or higher) operating system
- 200Mb of free HD space (>10Gb free HD space recommended for real time data saving)
How large are the wMicroTracker files?
The wMicroTracker creates a folder for each experiment. The raw data for a 2-hour experiment is about 20 Kb. The Excel export file for such an experiment is about 15 Kb.
Can I control/change the temperature of my samples with the wMicroTracker?
The temperature in the wMicroTracker cannot be pre-set and will depend on the room temperature. However, it is very compact and can easily fit in your incubator if needed. The wMicroTracker does measure and report the temperature inside of the platform for the duration of the experiment.
Note: The wMicroTracker’s power source does not generate heat.
Can I put the wMicroTracker inside an incubator?
The instrument is permeable to gas, so you can use a CO2 incubator when appropriate. Be sure to reduce humidity in the incubator. When using humid chambers for very short experiments, use a maximum experimental timeline of 12 hours.
What type of liquid media can I use? Can I use bacteria?
96-wells experiments work with: liquid culture of E. coli OP50, CeMM, CeHR (with or without 10% skim milk). Which one to use will depend on your preference and your experimental requirements. Note for lifespan experiments: it has been reported that liquid culture extend lifespan compared to NGM.
How many worms should I put in each well to get accurate measurements?
- For L1 to L3: use round-bottom 96-well plates: 100-150 larvae
- For L4 and older worms in a flat bottom 96-well plates: 30-70 worms
For more information on this matter please check out this Tech Note.
Do I need to count the worms in each well? How do I get good results if I can’t pipet a precise number of worms in each well?
Pipetting worms is not as precise as dispensing same number of worms with a cell sorter or even manually. However, you can easily reduce the variability of your data by relativizing mathematically each group of wells to time 0 (referred to as “BASAL measure” in Simonetta et al.; 2007).
What brands of plates can I use with the wMicroTracker?
The wMicroTracker was designed to fit multiwell plates from Greiner. Plates from other brands may not fit in the proper plate adapters.
For 384-well plates, you can use:
- 384w COSTAR square shape
- 384w Microtiter round shape by Thermo Scientific
Should I use flat-bottom or round bottom multi-well plates?
The swimming activity of worms in U-bottom wells is generally higher than in flat bottom wells since the shape of the well causes the worms to accumulate exactly in the path of the laser beam which detects activity. In flat bottom wells, the nematodes are more spread out and interfere less often with the laser beam. For more information, refer to this Tech Note.
C. elegans Synchronizer
Preferred method is to use 0.5M NaOH and / or (40khz) small ultrasonic cleaner. The ultra-sonic cleaner will ensure the filters are truly clean, while speeding up the process of cleaning. This will typically also take care of scaling. It is not recommended to descale the filters using an acid solution. Using an acid will at a minimum cause coloring/stains on the filter and repeated use of acids will ultimately damage the filters.
Yes, you can use small volumes of a worm culture. Recommended is a minimum of 10 gravid adults including laid eggs, however larger volumes have also been successfully processed in a single batch.
Yes, each strain is synchronized in approximately 10 to 15 minutes. Please keep in mind that each strain will have to remain in the ‘Harvest Filter’ for a period of time (i.e. ‘Harvest Time’). In order to synchronize multiple strains and not have to wait for the Harvest Filter to become available again, it is advised to purchase an extra ‘Harvest Filter’ for each additional strain. This will allow extended harvest time for each strain.
In a typical setup, where one person is working with the Synchronizer System, one (1) stabilizing filter should suffice. The Stabilizing Filter is used in the first step of the protocol and becomes available as soon as its content is transferred to the Harvest Filter. Depending on the on the need to exclude cross-contamination, the Stabilizer Filter may need to be cleaned / rinsed before processing the next strain.
Unlike the traditional filters, the CES filters do not have a ’round’ hole or ‘semi-round’ holes (aperture) so nematodes cannot swim through. This design also prevent the filters from clogging when a user tries to filter a large amount of a mixed population of worms. The filter design requires a very tight margin tolerance and an absolute zero defect of every single aperture in order to get nearly perfect synchronization.
Stimulus Preference Chip
Do you need the ScreenChip system to operate the stimulus preference chip?
The Stimulus Preference Chip is a standalone chip. You do not need a ScreenChip System in order to use it.
What are the minimal requirements for set-up?
To operate the stimulus preference chip, you will need:
- (4) 10 mL fluid-reservoir syringes with one-way stopcock and 20G needle stubs
- (1) 5 mL syringe with 17G blunt needle stub
- (4) PE/6 polyethylene tubing
- (3) PE/9 polyethylene tubing
- (4) 1.0 mm OD SS tube
- (3) 1.5 mm OD SS tubes
- (1) stereomicroscope
- (2) vacuum sources
Please reference the Stimulus Preference Chip User Guide for more information on the setup
How do you load the chip?
- Pick a worm to an unseeded agar plate
- Wash it with a drop of buffer, and allow it to crawl away from the drop
- Capture the worm in the end of the tubing of the loading syringe and insert it into the worm port
- Gently expel fluid until the worm is visible the near worm clamp.
- Wait a moment until the worm’s swimming motions cause its head to be pointed toward the clamp
- Push the worm midway through the clamp.
- Immediately activate the worm clamp vacuum. A well-positioned worm will exhibit sinusoidal movements of 1/4 to 1/2 wavelength at approximately the frequency of crawling on an agar surface.
- Open stopcocks 1 and 3, and activate the outlet vacuum to run the buffer solution through chip. At this point you may wish to record baseline behavior.
For more information on loading and setup, please reference the Stimulus Preference Chip User Guide
How long does it take to load the chip?
It takes about 5 min to load the chip with testing fluids at the start of an experiment. It takes about the same amount of time to wash and load a worm.
Does it need to be taped to bench for loading?
We recommend viewing the chip on a stereomicroscope. Taping the chip to the microscope stage is necessary to keep it from moving as the stopcocks are adjusted and while worms are inserted and removed.
How many worms can you load at a time (is there a waiting area like for the ScreenChips)?
For more consistent behavioral results from worm to worm, we recommend loading only one worm at a time into the chip. This is because there could be progressive changes in metabolic state as worms in the chip wait for their turn to perform.
How does preparation of the worm differ between the ScreenChip and the preference chip?
The main difference is that worms are washed in bulk for the ScreenChip, but individually for the Stimulus Preference Chip.
How do you load the stimuli?
Stimuli are loaded into syringes the feed into the chip. Please reference the Stimulus Preference Chip User Guide for further details.
What stage of worms does the stimulus preference chip accommodate?
The stimulus preference chip is designed to accommodate 1 day old adults
What type of stimuli can the chip deliver?
The Stimulus Preference Chip can deliver any water soluble compound as a stimulus in any of the four inputs. The chip can be operated in spatial or temporal modes:
- In the spatial mode, the solutions in the left and right channels are different. For example, the left and right solutions could be NaCl at different concentrations, yielding a step-wise concentration gradient. Or, the left and right solutions could contain the same concentration of NaCl and KCl, respectively, to measure the worm’s preference for Na+ and K+ ions.
- In the temporal mode, the worm experiences a sudden shift between two stimuli that are each delivered symmetrically. For example, the left and right channels could first contain a solution low in NaCl that is then switched to a solution high in NaCl to measure the worm’s response dynamics
How many worms can I screen with one chip?
If solutions are well filtered to prevent the chip from clogging (e.g., 0.2 um pore size), there is essentially no limit on the number of worms that can be tested in a single experimental session (e.g. one day in the lab). The chip can often be reused on subsequent days if it is cleaned and dried according standard procedures described in the Stimulus Preference Chip User Guide. This procedure works best when inorganic compounds are used as stimuli. In the case of organic odorants, there is the concern that these are retained by the chip to an unknown degree after cleaning. In this case, it is safest to use a new chip between daily sessions. A similar concern exists when bacteria are used as stimulants.
What type of data can be acquired with this chip?
The chip is designed primarily for the collection of behavioral data in several forms. These include the percentage of time the worm’s head resides on the left or the right side of the chip, and mean head angle in the chip (measured with respect to the worm longitudinal axis of the body in the worm clamp).
How can I analyze the data obtained from the Stimulus Preference Chip?
Video recordings of worm behavior (30 frames/sec) can be analyzed in MATLAB using a custom routine to compute head angle θ in each image.
- Frames mask and threshold frames to obtain an image of the worm.
- Use a skeletonization procedure obtain the centerline of the worm.
- Starting at the position of the restraint, traverse the centerline to find the tip of the head, defined as the point furthest from the restraint.
- Initiation of reversals can be scored manually by an observer who was blind to experimental condition. Reversal behavior was defined as propagation of the undulatory wave in the posterior to anterior direction as previously described.
Can I recover my worms?
How long can I keep the worm “immobilized” in the chip?
Yes, worms can be readily flushed out of the chip and recovered at the end of each experiment.
You can keep the worm trapped in place for up to 20 minutes in the Stimulus Preference Chip.