Let’s begin with how droplet ejection works. A beam of acoustic energy is focused onto a surface of a liquid, and because the acoustic energy does not transmit through the surface to the air it is then instead reflected. Because of this reflection the momentum of the beam is transmitted to the liquid surface which moves. With enough energy the surface will move far enough that the surface will wrap back around and create a droplet that escapes from the surface and moves away. There are three parameters that control this phenomenon, the amount of pressure on the surface, the size of the area of pressure on the surface, and the amount of time that this pressure is applied to the surface. A calibration file controls these parameters in order to produce the same volume droplet no matter how much liquid is in the well.
A wellplate will interfere with the acoustic beam. The material on the bottom of the well may absorb or reflect acoustic energy. The walls of the well may block a portion of the beam changing the amount of pressure and the size of the pressure area making it to the surface of the liquid. Therefore it is clear that thicker wellplates with smaller diameter wells will require more energy.
In the ATS system a calibration file is simply a look-up table. The ATS measures the position of the liquid surface and looks up what parameters are needed to generate a given drop volume. These parameters are the focus, the amplitude and the duration of the acoustic signal.
A calibration file is made using the provided software. This software takes a collection of data generated by the ATS and uses it to generate a calibration file for the ATS. When used properly this software is able to generate a calibration file that may be used to generate droplets of a given volume regardless of liquid level. By looking at the resulting calibration files one may compare the effects of a parameter.
The data that is used to generate these calibration files are three separate “well draining” processes. A “well drain” consists of repeatedly dispensing from the same well. For each dispense the liquid level or amount of liquid remaining in the well is measured. And, by measuring this both before and after a dispensing, this information is combined with the three parameters needed to generate the droplets and an accurate calibration is created for a given droplet volume. Figure 2 shows a collection of these calibration files used to generate a 2nl droplet for four different plates. These plates were chosen to be compared with each other because they are made out of the same material and they have the same well bottom thickness.
Therefore, the only difference between these plates is the diameter of the well. The first wellplate has a large well with a diameter of 3.2mm. The 2nl calibration for this wellplate is a relatively flat line of around 200 burst. The slight slope of the calibration is due to the change in acoustic system as focuses near the top of the well to the bottom of the well. This is the baseline well, a large well where the walls do not interfere with the acoustic energy. In addition to this well are three additional wells with successively smaller diameter wells have calibration files made. It is interesting to compare these calibration files with each other. The differences between them reflect the effects of the introduction of well walls on the acoustic beam.
The ATS is a very versatile machine with the capability to explore new ways to use acoustic energy for dispensing liquids. Understanding how it works enables one to know what to expect as parameters are changed. Knowing what to expect enables one to imagine what new uses the ATS could provide.
For more information on ATS Wellplate Calibrations, contact us at firstname.lastname@example.org.
Information Provided By Michael Forbush, PhD of EDC Biosystems.