In this chapter, I aim to provide a short conclusion and future experimental works, with respect to the techniques developed in each of the chapters.
9.1.
Dual optical tweezers
Figure. 9.1 shows a sequence of images (13 frames at 0.04s apart) of two 1 µm spheres held in two separate optical tweezers at around 15 µm apart. One of the trapped sphere (right) is broken down using the nanosecond laser. Residual bubble formation can be seen in frames 4-8. The second trapped particle (red arrow) is remained held in place after the LIB event. The figures are processed with help from Dr Y.Arita.
Conclusion - In chapter.2, I have list out the main design considerations required to construct a semi-automated dual optical tweezers system on a commercial inverted microscope system (Nikon TE 2000). The key equipment considerations in that chapter include the choice of laser and microscope objective lens. The important design considerations include the design of a dual beam optical tweezers system using a Mach Zehnder interferometry setup and beam steering optics with acousto optical deflector. Using the procedures describe in chapter.2, it is possible to build a high resolution optical tweezers for probing of biomechanical forces.
Future works – Using a dual beam optical tweezers system, it is possible to study both long and short range interactions between two particles held within each of the optical tweezers. The setup illustrated in figure 2.6 has now been used to study long range pressure waves during laser induced break down. Laser-induced breakdown (LIB) occurs when a high enough laser power density is absorbed by a given medium. Typically, a Q-switched (nanosecond) laser is used. The process of the LIB can lead to plasma formation, emission of an acoustic transient, which is followed by generation of a cavitation bubble [1]. The cavitation bubble can create a significant
pressure waves propagating in the medium can increase the membrane permeability of cells. In the current study, I have incorporated, via the epi-fluorescence port, a frequency doubled Q-switched Nd:YAG laser 532 nm with 1 ns pulse width carrying 1 mJ (Elforlight Ltd., Model SPOT) that is co-aligned onto one of the optical tweezers. Using one of the optical tweezers, I hold a single microparticle (1 µm in diameter) and localised the LIB on the trapped sphere. Using the dual optical tweezers system, the second particle is held at a distance and is used to measure the extent of the pressure being induced away from the LIB trapped sphere. The preliminary measurement of the pressure wave is to be covered in the future works pertaining to chapter.3. In the current system, there are two optical tweezers: one static and other steerable in the transverse directions. Both tweezers can be steered in the axial direction. The current dual optical tweezers system can be converted into a fully automated system by separately building lateral and axial steering devices into each of the beam path. In other words, the ideal system allows steering of two trapping beams in both the lateral and axial direction independently. This step will increase the flexibility of the position of a second probe particle in the experiment shown in figure 9.1.
9.2.
Back focal plane interferometry
Figure. 9.2 Position displacement from the probe microsphere (left particle figure 9.1 indicated by the red arrow). From the analysis of the particle position (before LIB (a) and after LIB(b)) shows that there is large displacement of around 400 nm during the blast and a secondary displacement of 100nm before returning back to the stable trapping position. The time frame that the recorded displacement occurs is shown in c, which is 0.3 ms. The figures are processed with help from Dr Y.Arita.
Conclusion – In chapter.3, I have introduced the techniques that have been used to measure the position and stiffness of an optically trapped particle. The main instrument used in this chapter is achieved using the BFP interferometry setup and a quadrant photodiode (QPD). I first use a nanopositioning stage so as to measure the change of intensity (interference patterns at BFP) with respect to the particle positions. The linearity region, where the change of intensity versus particle position is linear, is used as a calibration scaling to directly assess the position of a particle in an optical trap. For the calibration of the trap stiffness of the optically trapped object, there are two
methodologies used i.e. time and frequency domain. I then performed the two analysis method on a single trapped particle to assess the suitability of the methods. From the result, it appears that the frequency method can provide a more reliable and easier assessment of the trap stiffness. Using the frequency analysis method, I measure the variation of stiffness with respect to increasing optical power and axial trapping height. Using the Boltzmann distribution (particle position histogram over time), it is possible to directly plot the actual optical potential energy of the trapped particle at different optical powers. Finally, the calibration technique is used to calibrate the AOD steered optical tweezers. The optical tweezers system can experimentally measure particle displacement of up to 9 nm and mechanical force of up to 0.66 pN.
Future works –The framework of optical force measurement scheme provides unprecedented access to micro-mechanical effects in a non-invasive manner. In chapter.1 section 1.5.2, I have discussed on the use of the BFP interferometry system for the measurement of biomechanical forces of cells. In current work, I have extended the use of the BFP system onto the measurement of laser induced pressure waves. Current techniques on measuring these pressure waves requires the indirect imaging of free floating tracer particles with video tracking technique or the measurement of the expansion of the residue bubble formed after the breakdown. These techniques lack deterministic measurements of the actual forces that is important to assess the magnitude of the breakdown. Considering the use of the dual optical tweezers in section 9.1, I can directly use the BFP interferometry system and measure the displacement of a probe microsphere. The probe microsphere is held stably in an optical tweezers and positioned at a pre-determined location from the particle is being broken down. Based on the measured displacement (using the conversion table in table 3.1) in figure 9.2 b, c, I estimated that the pressure induced on the trapped particle is around 182 Pa. This is around an order of magnitude smaller than currently measured pressure based on the LIB of bulk material, which is reasonable. In figure 9.3, I showed the corresponding power spectrum density graph before and after the LIB event.
Figure. 9.3 Power spectrum of the position displacement from the probe microsphere (left particle figure 9.1 indicated by the red arrow). Just after the LIB event, there are a range of low frequency signal (f <100Hz). These fluctuations could be due to the expansion/contraction of the bubble formation after the LIB event. The figures are processed with help from Dr Y.Arita.
Although the results from the current data appear to be very promising, there are still some ambiguities that need to be resolve:
1) Issue - The bubble formation itself can affect the BFP interferometry measurement. This is because of the changes in the refractive index of the medium.
Possible solution - Use the probe beam, without any trapped probe particle, can be used to measure the background fluctuation signal during and after the LIB event. This can help indicate the background signal and remove any undesirable effects during a single measurement event.
2) Issue - The repeatability of the blasting event seems to be dependent on the particle size and axial trapping height, available on the particle to create a breakdown.
Possible solution – Need to repeat the data over a range of particle sizes and axial trapping height.
Besides this, I have constructed the QPD detection system onto four different optical trapping setups. In addition to the optical design, the list of softwares (stage controls (Prior scientific stages, Marzhauser stages), QPD (Hamamatsu Photonics) and PSD(Pacific silicon sensor) meant to convert the position data from the BFP interference into trap stiffness measurements have been written and designed on each individual platform. Finally, my software is currently used in a commercial optical tweezers system designed by Elliot scientific (UK).