Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0–100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.

Lyophilized disaccharides – the effect of water during storage

Nearly one fourth of new medicinal molecules are biopharmaceutical (protein, antibody or nucleic acid derivative) based. However, the administration of these compounds is not always that straightforward due to the fragile nature of aforementioned domains in GI-tract. In addition, these molecules often exhibit poor bioavailability when administered orally. As a result, parenteral administration is commonly preferred. In addition, shelf-life of these molecules in aqueous environments is poor, unless stored in low temperatures. Another approach is to bring these molecules to anhydrous form via lyophilization resulting in enhanced stability during storage. Proteins cannot most commonly be freeze dried by themselves so some kind of excipients are nearly always necessary. Disaccharides are commonly utilized excipients in freeze-dried formulations since they provide a rigid glassy matrix to maintain the native conformation of the protein domain. They also act as "sink"-agents, which basically mean that they can absorb some moisture from the environment and still help to protect the API itself to retain its activity and therefore offer a way to robust formulation. The aim of the present study was to investigate how four amorphous disaccharides (cellobiose, melibiose, sucrose and trehalose) behave when they are brought to different relative humidity levels. At first, solutions of each disaccharide were prepared, filled into scintillation vials and freeze dried. Initial information on how the moisture induced transformations take place, the lyophilized amorphous disaccharide cakes were placed in vacuum desiccators containing different relative humidity levels for defined period, after which selected analyzing methods were utilized to further examine the occurred transformations. Affinity to crystallization, water sorption of the disaccharides, the effect of moisture on glass transition and crystallization temperature were studied. In addition FT-IR microscopy was utilized to map the moisture distribution on a piece of lyophilized cake. Observations made during the experiments backed up the data mentioned in a previous study: melibiose and trehalose were shown to be superior over sucrose and cellobiose what comes to the ability to withstand elevated humidity and temperature, and to avoid crystallization with pharmaceutically relevant moisture contents. The difference was made evident with every utilized analyzing method. In addition, melibiose showed interesting anomalies during DVS runs, which were absent with other amorphous disaccharides. Particularly fascinating was the observation made with polarized light microscope, which revealed a possible small-scale crystallization that cannot be observed with XRPD. As a result, a suggestion can safely be made that a robust formulation is most likely obtained by utilizing either melibiose or trehalose as a stabilizing agent for biopharmaceutical freeze-dried formulations. On the other hand, more experiments should be conducted to obtain more accurate information on why these disaccharides have better tolerance for elevating humidities than others.

Tuning and stability of a singly resonant continuous-wave optical parametric oscillator close to degeneracy

Wavelength tuning and stability characteristics of a singly resonant continuous-wave optical parametric oscillator (cw OPO) in the proximity of signal-idler degeneracy have been studied. The OPO is made singly resonant by using a Bragg grating as a spectral filter in the OPO cavity. The signal-idler frequency difference can be tuned from 0.5 to 7 THz, which makes the OPO suitable for cw THz generation by optical heterodyning. The operation of the OPO within this singly-resonant regime is characterized by a strong self-stabilization effect. A gradual transition to an unstable, doubly-resonant regime is observed for a signal-idler detuning smaller than ~ 0.5 THz.

Water quality estimation by optical remote sensing in boreal lakes

In lake-rich regions, the gathering of information about water quality is challenging because only a small proportion of the lakes can be assessed each year by conventional methods. One of the techniques for improving the spatial and temporal representativeness of lake monitoring is remote sensing from satellites and aircrafts. The experimental material included detailed optical measurements in 11 lakes, air- and spaceborne remote sensing measurements with concurrent field sampling, automatic raft measurements and a national dataset of routine water quality measurements from over 1100 lakes. The analyses of the spatially high-resolution airborne remote sensing data from eutrophic and mesotrophic lakes showed that one or a few discrete water quality observations using conventional monitoring can yield a clear over- or underestimation of the overall water quality in a lake. The use of TM-type satellite instruments in addition to routine monitoring results substantially increases the number of lakes for which water quality information can be obtained. The preliminary results indicated that coloured dissolved organic matter (CDOM) can be estimated with TM-type satellite instruments, which could possibly be utilised as an aid in estimating the role of lakes in global carbon budgets. Based on the results of reflectance modelling and experimental data, MERIS satellite instrument has optimal or near-optimal channels for the estimation of turbidity, chlorophyll a and CDOM in Finnish lakes. MERIS images with a 300 m spatial resolution can provide water quality information in different parts of large and medium-sized lakes, and in filling in the gaps resulting from conventional monitoring. Algorithms that would not require simultaneous field data for algorithm training would increase the amount of remote sensing-based information available for lake monitoring. The MERIS Boreal Lakes processor, trained with the optical data and concentration ranges provided by this study, enabled turbidity estimations with good accuracy without the need for algorithm correction with field measurements, while chlorophyll a and CDOM estimations require further development of the processor. The accuracy of interpreting chlorophyll a via semi empirical algorithms can be improved by classifying lakes prior to interpretation according to their CDOM level and trophic status. Optical modelling indicated that the spectral diffuse attenuation coefficient can be estimated with reasonable accuracy from the measured water quality concentrations. This provides more detailed information on light attenuation from routine monitoring measurements than is available through the Secchi disk transparency. The results of this study improve the interpretation of lake water quality by remote sensing and encourage the use of remote sensing in lake monitoring.