HomeTroubleshooting Guide

Visual Synchronizer

  • Error: ‘Camera not being triggered error’
    • Check that the camera-to-amplifier cable (USB-C to a circular connector on the camera) is firmly connected.
  • Error: ‘Dropped Frames error’
    • Reduce the camera framerate in the Camera settings dialog box accessible by clicking on the Settings button in the right pane. If you need to use a high framerate, select a Region of Interest (ROI) by adjusting the blue rectangle to the smallest possible area and press the Zoom in button. NemAcquire is optimized to only record and process the ROI portion of the captured images, which allows you to get a higher framerate.
  • Unable to focus on a worm
    • If the image is in focus through the microscope’s eyepiece but not on the screen, adjust the adapter’s length and / or the stage height to bring the ScreenChip into focus.
  • Cannot increase the exposure time
    • The exposure time is limited by the framerate.  For example, a framerate of 25 fps results in a maximum exposure time of 40 ms. To increase the exposure time, lower the framerate in the Camera settings dialog, which is accessible by clicking on the Settings button in the right pane.

Software

  • The software I downloaded will not launch. Why?
  • The LED light on the amplifier does not turn on when I open NemAcquire.
  • I do not see any signal in NemAcquire.
  • I have placed a worm into the chip, but I do not see a pumping signal on NemAcquire

Loading your worms

  • My chips get clogged. What should I do?
  • How can I load my worms in the chip?
  • What vacuum source can I use with the ScreenChip system?
  • What size chip do I need?
  • The worm’s tail and head are moving when it is placed in the recording channel. Does it affect the EPG data?
  • The worms keep sliding forward or backward into the channel

Stimulation of pharyngeal pumping

  • Do the worms pump in M9?
  • What type of serotonin can I use to stimulate pumping?
  • I want to feed the worms inside the chip. How do I proceed?
  • I want to use drugs in the chips. What do I need to know?

Reducing noise

  • What is a normal level of electrical noise?
  • What does a normal EPG trace look like?
  • My recordings are noisy. What can I do to resolve this?

Acquiring data

  • How do I record the EPGs?
  • I have placed a worm into the chip, but I do not see a pumping signal on NemAcquire
  • The worm signal is small or not appearing as expected; why is this?
  • The pump frequency is low (below 3Hz); why?

Software

  • The software I downloaded will not launch. Why?
  1. Make sure that your computer uses an updated operating system (Windows 10 or up for PCs and Mac OS X or up for Mac computer. You will not be able to acquire data if your computer OS is not updated.
  2. Check that you have downloaded the correct file for your operating system. If you are using a Windows computer, you need to download the software files noted “(PC)”.
  • The LED light on the amplifier does not turn on when I open NemAcquire
  1. Check that the amplifier is connected to your computer and to the dock. The green Led lamp will turn on when the amplifier connects to the software.
  2. Make sure that your computer uses an updated operating system (Windows 10 or up for PCs and Mac OS X or up for Mac computer. You will not be able to acquire data if your computer OS is not updated.
  • I do not see any signal in NemAcquire.
  1. Check that the amplifier is connected to your computer and to the dock. The green Led lamp will turn on when the amplifier connects to the software.
  2. Is the chip filled with saline buffer?
  3. Is there a worm in saline buffer located in the main recording channel?

Loading your worms

  • My chips get clogged. What should I do?

Look under the microscope to identify the nature of the clog.

How to prevent clogs:

  1. Thoroughly rinse your worms in buffer (i.e. M9) before use (see protocol here)
  2. ALWAYS check the epitube under a microscope after rinsing to ensure that you have removed all bacteria, dust and fibers. Use a pipette to remove visible fibers if necessary.
  3. Do not use lint-producing material (i.e. KIM wipes, paper towels)
  4. Do not insert the inlet tubing too deeply into the chip. This can cause small pieces of PDMS to break loose.

How to unclog your chip:

  1. Grab your syringe kit (5ml Syringe + 17G needle stub – included) and fill it with your experimental buffer
  2. Remove the inlet tubing from the epi-tube and connect the syringe.
  3. Gently push the plunger to push the clogging body toward the outlet using positive pressure. Do not push too hard, or you might break your chip!
  4. Repeat steps 1-3 using water to rinse out the saline.  Let the chip air dry, then place it back into the original packaging for storage.
  5. This method often helps to remove soft debris (e.g., clumps of eggs and bacteria), however hard items (e.g., chunks of agarose or plastic) may clog the chip permanently.  If this method does not work, discard and use a new chip.

 

  • What size chip do I need?

Please refer to the ScreenChip selection guide

  • The worm’s tail and head are moving when it is placed in the recording channel. Does it affect the EPG data?
  1. Check that you are using the correct ScreenChip size: ScreenChip selection guide
  2. If the worm fits in the channel and is unable to swim completely freely, then you are able to collect pharyngeal pumping data. If the worm can swim without touching the walls of the channel, you may need to use a smaller chip.

The chips are tapered which allows the worm to enter the channel before being trapped at the constriction. Because the worms head and tail are narrower than the rest of the body, the can move freely, allowing the worm to feed. This movement will not alter the acquisition of your data.

  • The worms keep sliding forward or backward into the channel. Does it affect my data? What should I do?

As long as the worm’s pharynx is located between the electrodes of the chip, you will be able to detect a clear signal. Please note that the recording channel is tapered to allow worms of varying sizes to fit. This means that as the worm slides within the recording channel, the seal (contact of the cuticle to the walls of the channel) will change, thereby changing the amplitude of the signal detected. The other parameters recorded will not be affected.

Stimulation of pharyngeal pumping

  • Do the worms pump in M9?

Day 1 N2 adults do not readily pump in the absence of a pumping stimulant. In saline buffer such as M9 or S-Basal, these worms typically pump between 0 Hz and 3 Hz. The variability of pump frequency within one population can be high and on average, the pump frequency of a N2 Day 1 worm population is below 1 Hz.

We recommend that you use pumping stimulants in the chip, such as serotonin or bacteria, in order to trigger pharyngeal pumping. Using a pumping stimulant will allow you observe a higher number of pumps, making the statistical analysis more stringent. In addition, since the pumps are sparse during a single recording, the variability of interpump intervals is usually high.

  • What type of serotonin can I use to stimulate pumping?

We use Serotonin hydrochloride for our in-house experiments and recommend that you use it as well. 10mM of serotonin in M9 will allow you to stimulate pharyngeal pumping to its maximum frequency (4.5 to 5.5 Hz in healthy young adult N2 worms). For your convenience, we offer pre-weighed aliquots of serotonin. See RediPrep™ Serotonin.

  • I want to feed the worms inside the chip. How do I proceed?
  1. To feed the worms within the ScreenChip, make sure that you use a consistent food source based on bacteria quality, number of cells/mL or OD600.
  2. Make sure that the bacteria completely resuspended in buffer, without clumps. This will allow you to stimulate pumping with bacteria without clogging your chip.

When feeding worms within the ScreenChip we recommend using freeze-dried bacteria. Freeze-dried bacteria ensures a consistent food source based on bacteria quality, number of cells/mL. See Instant freeze-dried OP50.

  • I want to use drugs in the chips. What do I need to know?

The ScreenChip is make in part of PDMS. PDMS absorbs drugs, especially hydrophobic small molecules. Once a drug has been run through a chip, the PDMS is contaminated. Thoughtful experimental design prolongs the useful lifetime of a chip; e.g., test low concentrations of drugs before higher concentrations.

Reducing noise

  • What is a normal level of electrical noise?

In a chip filled with a saline buffer and with no worm, the electrical signal should remain between 9uV and 20uV as displayed on the top left corner of NemAcquire.

  • What should the traces look like?

A standard EPG (electropharyngeopgram) signal is composed of a positive peak (corresponding to the excitation of the pharyngeal muscle) and a negative peak (corresponding to the relaxation of the pharyngeal muscle). Although the overall shape of the trace can change due to visualization parameters and inter-individual variability, we found that the quantifiable parameters are often consistent within a population.

  • My recordings are noisy. What can I do to resolve this?
    1. Disconnect the outlet tubing from the chip. After a couple of days of use, a biofilm forms within the outlet tubing, making it act as an antenna that generate high electrical noise. This problem can be solved by removing the outlet tubing before each recording. You can also opt to replace the outlet tubing once a week.
    2. The Powerline noise is generated by nearby electrical appliances (centrifuge, nutator, laptop charger, microscope lamp etc…). This signal is sinusoidal and has a frequency of 60Hz in the US and 50Hz in the EU. The powerline noise can be very high when a large equipment, such as a centrifuge or a nutator is functioning in the vicinity of the system. In such case, a simple notch filter might not be sufficient to clean up the signal. It is important to check the electrical noise before each experiment, to make sure that the electrical interference is minimal. A peak-to-peak amplitude of 20uV or less is an ideal condition for a clean, easy to analyze recording.
  1. Load the chip with saline buffer, no worms
  2. Make sure that the cables are not touching or obstructed
  3. Unplug your laptop charger
  4. Turn off the microscope lamp
  5. Unplug any appliance from the wall plug near your set-up
  6. Make sure that no large centrifuge is running in the same room or, if you are located near a shared wall, in the next room.
  7. If you still experience noise, pick up your dock and amplifier, keeping them connected, and watch the noise as you leave the room. If the source of noise is located in the set-up room, moving the system outside will eliminate the noise
  8. If you are still experiencing noise, make sure that you are using a new chip. Re-using the chips can lead to electrical noise as the electrodes get eroded.

Acquiring data

  • How do I record the EPGs?
  1. Make sure that you are using the latest version of your operating system (Windows 10, or iOS X 10.11)
  2. Download NemAcquire
  3. Connect your amplifier to your computer via USB2.0 or USB 3.0
  4. Open NemAcquire: You should see a signal appear
  5. Pre-load the chip with buffer
  6. Check that your electrical noise is below 20uV (top left corner of the grid)
  7. In the ‘File’ menu, select the recording folder – This is where your data will be saved
  8. Load a worm into the recording channel
  9. Click on ‘record’
  10. Fill-in the experimental data for your worm
  11. Stop recording and click ‘Save’.
  • I have placed a worm into the chip, but I do not see a pumping signal on NemAcquire

Check that:

  1. You are using the latest version of your operating system (Windows 10, or iOS X 10.11)
  2. The amplifier is connected to your computer:  The green LED turns on when opening NemAcquire and you see a white trace on the screen
  3. There is still a worm in the chip: Check under the microscope to verify that it did not slide out of the recording channel
  4. You have added a pumping stimulus in the buffer (i.e., 5HT, OP50): Without a pumping stimulus, adult N2 worms pump very irregularly (average frequency below 1Hz).
  5. The worm is alive: Is the worm moving its head and/or tail?
  6. The worm is pumping: Can you see the pharynx moving through the micros
  • The worm signal is small or not appearing as expected; why is this?
If the signal amplitude is small or not visible, there are several things you can do to.
  1. Removing the exit post tubing from the chip during recording can increase the signal-to-noise ratio.  fluid or bacterial growth in the exit tubing can act as an antenna. Make sure that yolu disconnect the outlet tubing from the chip before recording.
  2. Make sure the worm is sitting between the two electrodes in the chip channel.
  3. If the worm begins to slide out one end, the signal amplitude will decline or not be captured at all.
  4. The worm must be large enough for the channel. Worms that are too small for the chip will make a poor seal with the channel and the signal will be weak.
  5. Sometimes fluid or bacterial growth in the exit tubing can act as an antenna. Try removing the exit tubing during a recording, or replacing the exit tubing if problems continue.
  • The pump frequency is low (below 3Hz)
  1. Check that you are using a pumping stimulus (serotonin, bacteria). In the absence of pumping stimulus, a population of healthy N2 young adults pumps on average below 1Hz
  2. If you are using serotonin, make sure that you dissolve the serotonin right before your experiment. Use the serotonin within 2 hours of preparation. At room temperature, serotonin decays rapidly, leading to a decrease in efficiency, which will appear as a dip in the pumping frequency of your worms.
  3. If you use bacteria, make sure that you starve your worms (usually 2 hours) on an unseeded plate before your experiment. Animals that have not been starved tends to show a lower pump frequency, and an inconsistent pumping pattern. The ScreenChip system is very sensitive, therefore it is important to use consistent food source in terms of quality and bacterial concentration. When feeding worms within the ScreenChip we recommend using freeze-dried bacteria. Freeze-dried bacteria ensures a consistent food source based on bacteria quality, number of cells/mL. See Instant freeze-dried OP50
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