A protocol is a type of experiment defined by the instructions that the instrument has to perform. The minitweezers can control the position and the force of the optical trap with a feedback. A protocol is defined by the steps that the instrument has to automatically perform on a molecule. For instance, one of the most common protocols is a pulling experiment. It consists on moving the optical trap up and down at a constant rate so that the molecule undergoes cycles of stretching and relaxing. Another protocol is the constant force protocol, in which the molecule is held at a constant force. This is achieved by means of a force feedback that corrects the position of the optical trap to keep constant the force exerted on the molecule.
The firmware and software of the instrument was designed so that new protocols can easily be implemented. Indeed, most of the essential routines and functions that perform the elementary steps are available to the user. Thus, a new protocol does not need to be designed from scratch. Instead, users can gather pieces of already coded subroutines to create their own protocol.
Here we have to distinguish between two types of new protocols. The first one only involves coding the software and it is easier to implement. The second one is more demanding and, apart from the software, it requires to code the firmware of the PICs. The advantage of coding the PICs is the update frequency. Indeed, the software of the Mac can update the instrument at 60 Hz, while the firmware of the PICs, at 4 kHz. Depending on the requirements of the experiment, one type of protocol will be preferred over the other. Appendix D shows a detailed description of the steps to be followed to code a new protocol.
Figure 2.17 shows data obtained with five different protocols designed during the realization of this PhD thesis:
- Double trap pulling protocol.
- This protocol assumes that each counter-propagating laser is used as an independent optical trap. Therefore, two beads located at two optical traps are used to pull on a molecule. The traps can move simultaneously or one respect to the other (see Fig. 2.17a).
- Oscillation protocol.
- This protocol produces an oscillation of the force or the position of the optical trap. The frequency and the amplitude of the oscillation can be adjusted (see Fig. 2.17b). This is useful to study the stochastic resonance of single molecules .
- Force jump protocol.
- This protocol is a combination of two already existing ones. It consists in a pulling experiment followed by a constant force protocol at a different arbitrary force (see Fig. 2.17c). The misfolding dynamics of DNA hairpins can be studied with this protocol.
- Force ramp protocol.
- It is a pulling experiment in which the force applied to the molecule is increased at a constant loading rate. Instead of constantly changing the position of the optical trap, this protocol continuously increases the force by running a force feedback. The comparison between a force ramp protocol and a regular pulling protocol gives relevant information about the thermodynamics of small systems. This protocol was designed to carry out the work described in chapter 6.
- Pulling protocol coded in the firmware.
- It is a standard pulling protocol in which the molecule is stretched and relaxed by moving the trap up and down. The advantage is that the update frequency of the PICs is 4 kHz and the position vs. time does not exhibit steps at high pulling speeds (see Fig. 2.17d).
New designed protocols. (a) Unzipping of DNA with a double trap pulling protocol. The FDCs of the traps (red and orange) are measured simultaneously. Except for thermal fluctuations, the forces are equal and opposite. (b) Oscillation protocol applied to a DNA hairpin. The force oscillates between 14 and 15.4 pN (red curve) according to a square wave of frequency 0.4 Hz. The position of the optical trap (blue curve) is continuously adjusted by the force feedback algorithm. (c) Force jump protocol applied to a DNA hairpin. A pulling protocol of pulling rate 30 nm/s is followed by a constant force protocol of force 14.5 pN. (d) Position vs. time curves of two pulling protocols at a pulling rate of 950 nm/s. The blue (magenta) curve shows the output of the protocol performed by the PIC (host computer).