On this page, standard tasks related to running DoD experiments are collected with step-by-step guidance. This document is intended for users and staff alike. However, some of the tasks and solutions shown here are far beyond standard use cases. Just because it is outlined how to do it, does not mean YOU should do it. Contact your instrument scientist or SED POC before doing invasive interventions (Hint: If you need a screwdriver or hex-key, it is invasive).
Overview over the system
The Drop-on-Demand sample robot consists of a few components, which will be referenced later on in this guide. Therefore, the key components are shown here:
The sample robot
the nozzle holder sits on a large motorized x-y-z stage.
Sample holder in base configuration without PDCs
Pump tower with syringe pumps (top) and peristaltic pump (bottom) for wash station
The syringe pumps are pumping system liquid (bottle with blue cap) to the back side of the PDCs.
Auxiliary stations in the base version
From the back to the front on the left: Camera, washstation, bath, eppendorf tube holder, dab station, empty slot, LED for illumination.
On the right, we see the temperature controlled well-plate holder without an inset.
Fluid manifold
This is where the PDCs are connected to. The back side is connected to the system liquid. The channels are numerated 1-4, from upper left to lower right.
The E-box (motor controller)
The large grey box is the controller for the robot, with the windows PC that runs the software shown in the second image on the lower right. It plugs into the network for connection.
SciPulse units channel 1 and 2
These are the electrical drivers that are controlling the Piezo-signal to channels 1 and 2, respectively. Channel 3 and 4 are driven from the E-box, using a PU pico unit integrated inside.
Getting started: Standard tasks
Emergency stop: Aborting unwanted motion
In case of any unwanted or dangerous motion of the machine, the emergency-stop can be pushed. It is a red button on a yellow box, and located close to the robot. After it is pressed, the power to the axis is cut by a safety relay. In order to reset the system again, the device needs to be restarted afterwards with a full power cycle, since otherwise some of the components remain in a faulty state. Also, the emergency button needs to be pulled up again (into a de-pressed state).
In the right image, the two safety relays can be seen (SS1 and SS2). They are located inside the E-box (large controller unit) and can be accessed by unscrewing the front panel. Sometimes they can stay in a faulted state. Then it is useful to check electrical connections. If the relay stays in a faulty state, it can help to rotate the knob at the top (not shown in image) using a flat screwdriver out of its position and back into the previous position to bring it back manually.
There is also an electronic stop button in the software interface (shown in red) which stops axis motion. It will stop any motion of the axis without bringing the system into a faulted state, but since it is software-reliant, it is less fail-safe.
System startup
- Power up the E-box by rotating the red button at the front side into its on position. Then, press the start button (transparent button next to the green light). Now, the PC should start itself. If not, go to the back side of the E-box, and klick the power button of the PC.
- Wait for windows to be fully operational and responsive. The password can be obtained from one of the instrument staff.
- Power up the sciPulse units afterwards (important, as the com-port buffer can get messed up if not done, and communication with the controller fails) by switching the power buttons at their front panels.
- Start the software, which has either a hyperlink on the desktop or is located in the program folder on the desktop. The name is "sciBeamliner" with the latest version number. Always run the latest software version.
- Upon startup, the software asks for a user profile. Use the appropriate one for the given configuration. Often, this would be sciPulse
- Before klicking the initialization button, make sure that the robot can travel freely. It will move to its limit switches (all the way up, all the way back, all the way to the right when standing in front of the robot. It could collide if the path is not clear.
- After the stages are initialized and the robot has recognized all components (sciPulse units, pumps, chiller - if connected), the software is in its base state.
Priming the system before first use of the day:
Before using the DoD system for the first time of the day, it is important to properly prime the system.
- Degas the system liquid
- Fill the system liquid bottle with filtered DI water. The fill height has to be chosen such that the liquid level has approximately the same level as the tip of the PDCs.
- Place the system liquid inside the ultrasonic bath and connect the pump adapter to the head.
- Turn on the pump and make sure that the pump creates a lower pressure inside the bottle, i.e., that the white adapter piece is held in place tightly by the suction. Otherwise the degassing will be unsuccessful. The pressure reading on the pump should decrease.
- Turn on the ultrasonic bath, and let the system run for 30 minutes.
- To speed up the process, you can rotate the system liquid bottle manually and submerging it into the ultrasonic bath. This will promote degassing. In the end, nearly no new air bubbles should form.
- Turn off the pump and disconnect the bottle. To release the lower pressure, the connector piece shown below can be removed, which will release the pressure.
- Place the system liquid in the appropriate secondary containment next to the robot (fill height should match nozzle position!) and screw on the cap with the 4 lines. Make sure that all 4 lines are submerged below the water level.
- If in standard configuration, run the "prime run". If not, follow the basic steps below:
- Load the PDCs into the sample holder, without connecting them. Activate all selected nozzles in the software. If you don't do this, the nozzles will not be moved down with the Stinger unit, if equipped, and the liquid will be flushed into the unit, leading to short circuiting of the PDCs
- Connect the wash bottle to the fluid manifold
- Start flushing the lines with large amounts of water (wash lines (1min) as well as syringe pump (>5ml @ 40ul/s)
- Align the PDCs to the camera station with their respective offsets. Save positions and initial droplet ejection parameters
- Disconnect the wash bottle
- Start pumping liquid out of the syringe pumps (2ml @10ul/s). Once the first droplets has left the fluid manifold, screw in the correct PDCs into the manifold. Pay attention to the right channel choice.
- After flushing the syringes, the nozzles should be able to drop nicely. Check that each nozzle is working well. If not, perform basic debugging steps (see #Getting stable ejection)
Swapping nozzles:
Standard setup <Images!>
- Disconnect all PDCs from the fluid port.
- Loosen the 4 thumbscrews around the nozzle holder.
<Image of the standard holder> - Slide off the nozzle holder and place it on a table. Be careful not to touch or break the nozzles at the front.
- Slide out the acrylic glass
- Remove the 2 holding pieces.
- Slide out the nozzles you want to remove, and store them into their respective packages. Match the IDs to the package!
- Insert all the nozzles you would like to use by sliding them into the respective positions and pushing them as far down as possible. If there is friction / an edge blocking the motion, rotate the PDC around its axis until it slides.
- Insert the lower holding piece by clamping the springs with it down.
- Insert the upper piece
- Slide the transparent plastic back in place. The side is groved at 45 degree, so it will only work in one direction.
- Slide the holder onto the 4 holding posts, until it is in place. Be careful that the tubing of the PDCs are not dangling around to accidently touch or break the nozzles.
- Tighten the 4 thumb screws.
- Prime the nozzles by the steps outlined above.
Stinger setup
The Stinger allows the use of the 4 nozzles in channels 1-4.Each nozzle is installed in an individual sliding holder.
- Bring the nozzle that is supposed to be uninstalled in the up position by klicking the green button in the software. Before uninstalling, it needs to be in the up-position (greyed out). Since always one nozzle has to be selected, you can select one of channels 5-8, since there is no physical connection here, in case all nozzles are supposed to be uninstalled.
- Open the front door of the Stinger by lifting it up slightly and turning it sideways.
- Disconnect the PDCs that are supposed to be changed.
- Take off the hook of the PDC from its metal guide, while holding the PDC in place. This might require a tweezer, depending on your finger size.
- Slide the holder upwards, while keeping it as close as possible to the vertical orientation. Tilting it too strongly will result in the nozzle tip clipping on the lower end.
- Once the holder is high enough for the nozzle to come out without clipping on the lower side, take it out.
- The holder consists of 2 parts. Unclip the upper small part that holds the spring in place.
- Slide out the PDC and store it in its container.
- Slide in new PDC.
- Slide it in so far that the piezo (black piece) is nearly invisible.
- Bring the spring into the right position, so that it rests on the lower side (closer to the nozzle). Take the small holding piece, and clip it tightly onto the PDC. Make sure that it sits flush and holds the spring in place.
- Slide the holder back into the Stinger in the most vertical orientation.
- Hook it back onto the guide.
- Close the Stinger door, and connect the PDC.
- Prime the exchanged nozzle.
Colliding droplet setup
- Take out the connectors of the nozzle holder
- Disconnect the fluid port of the nozzle
- Loosen slightly the screw of the holder connecting it to the rotation pad while holding it in place (in might slip off otherwise).
<Image of holder with one electrical connector undone>
- Slide it upwards and take the holder off. Place holder on intermittent holder so that the inserted nozzle is protected
<Image of sliding off holder>
- Take new nozzle holder with nozzle and slide it in place. Be careful not to slide it too far so that the nozzle collides with the second nozzle.
- Tighten the screw
- Connect fluid port
- Connect electrical connectors and make sure that they are sitting securely in place.
<Image of insertion of nozzle holder>
- Perform priming steps for new nozzle, as well as alignment steps if in colliding droplet mode.
Colliding droplets: Installing the alignment target
Colliding droplets: Priming the injector (Task ColDrop_Prime_UP)
- If required: Replace the system liquid and wash liquid. Make sure that liquid level is about the height of the nozzle tip + x (determined by overpressure in the enclosure that works against the hydrostatics). The system liquid should be degassed beforehand
- Connect the flush bottle to the manifold.
- Wash ca 10ml @ 50ul/s = 200s through the lines
- Move the injector to the wash station up (via waypoints (safe min(y), min(x), min (z)), (safe min(y), max(x), min (z)), (safe wash(y), max(x), min (z))
- Disconnect the wash bottle.
- Turn on the pump @10ul/s, wait for droplet to form at connector, wet connect the PDCs
- Turn on the wash pump
- Connect the PDCs
- Move PDCs inside the wash station via waypoints.
- Actuate the piezos
- Tasks ColDRO_prime_nozzle_up_part1 – part4
Colliding droplets: Finding spatial and temporal overlap
Nomenclature: The nozzle in the higher position when the robot head is rotated is channel 1 or “up”, the other nozzle is channel 2 or “down”. The voltage is provided from the
- Test that each nozzle is primed
- Save the coordinates of the nozzle up and nozzle down in the position list.
- The approximate position of the collision point is given from geometrical considerations. Example: For 90 degree collision angle:
- Height coordinate is the mean between the nozzles.
- The horizontal coordinate is given by x_mean – delta_z/2
- Turn on both nozzles by clicking ctrl + both nozzles and then the start button in the UNtriggered mode. Then, vary the LED delay for each nozzle individually, until the shape of each droplet is overlapped. This will allow to determine the required trigger offset between the nozzles. The difference between the LED delay is the difference of the triggers required.
- Adjust the trigger of the channel with the faster LED timing by the difference between both LED signals.
- For imaging, use the LED timing of the slower channel now; By this LED delay + x, you should observe collision.
- When this has been done, turn over to the triggered mode and make sure that both droplets are here as expected.
- Bring the collision point to the sideview imaging. This should
Colliding droplets: Priming the sample aspiration stations
Tasks ColDRO_prime_sample_up_part1 and ColDRO_prime_sample_up_part2