The main goal was to automate manual patch clamping while retaining high quality data. A definition of a successful experiment encompasses the following parameters: a true GigaSeal of more than 1 GOhm, a series resistance of less than 5 MOhm in whole-cell mode, a continued total resistance between 0.5 and 1 GOhm, and a duration of these parameters of at least 15 min.
Given a 3 or 6 channel robot, a data volume of around 140280 data points per day can be collected, given a twofold compound application per FlipTip and a recording time of 20 min.
Further, the FlipTips have a much lower stray capacitance (<10 pF) compared to chip-based systems, making them useful for studying fast voltage-activated ion channels.
The Flyscreen software uses an internal scheduler for intelligent robot control. This scheduler optimizes the throughput by maximizing the parallel execution of experimental steps. In essence, electrophysiological operations are performed in multiple recording channels while another experiment is using the robot arm on a different recording tip socket channel.
Similar to manual patch clamping, a task is finished or cancelled once a user-defined flag condition has been met or not. As an example, a seal segment is set to last 5 minutes to reach a user-defined seal resistance. If the value has been reached before the specified time period, the scheduler shifts all following segments forward in time. This is performed for every recording tip socket independently.
In panel A of Figure 4, a completed batch of 15 FlipTips is shown, and individual segments are depicted in different colors. Most notably are the red segments for RPip validation and control measurements (channel expression test), yellow seal segments, and green segments indicating compound application. Among the three tip sockets, it is evident that the segments do not all occur at the same time, reflecting the action of the scheduler. This batch took 1 h 40 min to be completed, and showed a success rate of 80% (seal and whole-cell formation).
Panel B of Figure 4 shows the successive blockade of the Kv1.5 potassium channel expressed in an LTK cell line by 5 mM 4-Aminopyridine, and panel C depicts the time course of a blockade shown in an online analysis window. The user can export all the data points in an online analysis window easily as ASCII values to construct dose/ response and I/V relationships. Several on-line analysis windows can be opened at the same time to analyze different aspects of the data trace. Thus a multi-parameter analysis can be performed with a single experimental run.
Panel D shows a FlyStat analysis over a time period of 18 days, revealing the success rate for valid FlipTips (RPip), seal and whole-cell state, and the recording time.