NIELS BOHR INSTITUTE

UNIVERSITY OF COPENHAGEN

OPTICAL

TWEEZERS

biophysical research group


Research

Biophotonics

Single molecule biophysics

Cellular biophysics

Model systems

Research interests

Biophotonics

Optical manipulation of metallic nanoparticles

We have shown that metallic nanoparticles such as silver or gold nanospheres or rods can be individually optically trapped using a CW infrared laser. If the particle is elongated, it aligns with the polarization of the laser.

The plasmonic interaction between the particle and the incoming electromagnetic field will cause the particle to absorb part of the incoming light. The absorbed light is dissipated as heat, thus creating a local heat gradient around the nanoparticle.

Using a lipid bilayer assay, we directly measure the temperature at the surface of irradiated metallic nanoparticles, which can easily increase by hundreds of degrees Celsius.

Focus aberrations, optimization

Dying HUVEC cells

In practice, it is nearly impossible to obtain a perfect focus of a laser beam as spherical aberrations are inherently present. Commercially available microscope objectives are nearly always optimized for visible wavelengths thus introducing significant aberrations in the infrared regime, were most tweezers setups work. The more aberrated the focus, the weaker the trap.

Spherical aberrations can be efficiently canceled, even deep into the sample, simply by changing the index of refraction of the immersion media. Or, if using a water immersion objective, by changing the dial to a thickness different from the physical coverslip thickness.

To quantify and remove noise we suggest including Allan Variance in the standard calibration.


Single Molecule Biophysics

In vitro: Physical properties of DNA and mRNA pseudoknots

In a living organism molecular motors, operating on nucleic acids, constantly force nucleic acid structures to twist, stretch, open or melt. Using optical tweezers we mimic the action of the molecular machinery and probe physical properties of DNA and mRNA structures.

The mechanical strength of mRNA pseudoknots was found to correlate with in vivo frameshifting efficiency. However, if the pseudoknot becomes too strong, ribosomes will stall within the structure. We are presently investigating the correlation between the pseudoknot structure and its mechanical strength and how this relates to framshifting efficiency.

Also, we are interested in how DNA behaves under tension and find that DNA twisting and stretching are intimately related and that DNA melts in a stick-slip fashion at the overstretching transition.

In vivo: Manipulation of individual membrane proteins

Fluorescent 'bird' from stained cell membranes.

By in vivo labeling of a protein (the lambda receptor) in the outer membrane of E. coli, we were able to uncover the motility of an individual receptor. The receptor performed a confined diffusion in the membrane.

Interestingly, the receptor's motility depended on the metabolic state of the microorganism: If the bacteria were depleted for energy, the motility ceased significantly. This is also true if the outer membrane was targeted by ampicillin, vancomycin, or specific antimicrobial peptides.

Hence, a metabolically competent bacteria spend energy to move its membrane proteins, possibly to facilitate transport through the receptor. We are presently interested in whether this is generally the case for other membrane proteins and cell types too.


Cellular Biophysics

Anomalous diffusion and ergodicity breaking within living cells

Diffusion is the most important transport mechanism within a living cell, where the small dimensions mean, that diffusion times are acceptable for cargo transport.

Stained membranes of S. pombe cells

We investigated the diffusion of lipid granules within S. pombe fission yeast cells. The diffusion was anomalous with subdiffusion dominant at small time lags. At larger time scales also confined motion, normal Brownian motion, and superdiffusion occurred as signs of biological processes. Interestingly, time average mean squared displacement analysis show that the temporal averages deviate significantly from the ensemble averages. Hence, the system exhibits weak ergodicity breaking. For lipid granules inside endothelial cells, only granules close to or inside the nucleus exhibit ergodicity breaking.

We continue our efforts to understand the complex transportation pathways inside living cells.

Cellular adhesion, spreading, dynamics of monolayers

Using endothelial cells lining the blood vessels as a model system, we investigated how a living endothelial cell adheres to the surface, how it spreads out and how it migrates into open space. The dynamics are relatively complex and modified in a non-trivial manner by addition of arachidonic acid, which basically causes the cell to lose its sense of direction. Hence, arachidonic acid has prospects to block angiogenesis and could possibly be used as cancer treatment.

As the natural environment for an endothelial cell is to be in a monolayer we are presently investigating the dynamics of entire cellular monolayers with special interest in how energy is dissipated and fingering (creation of new blood vessels) occurs.


Biophysical model systems based on lipid assays

Quantification of heating of irradiated nanoparticles using a bilayer assay

Lipids melted by heated gold nanorod. Fluctuations stems from rotating laser polarization.

If the plasmon resonance of an irradiated metallic nanoparticle coincides with the wavelength of the irradiating light absorption and associated dissipation of heat can be rather substantial. As the intensity distribution within a focused laser beam typically is rather complex due to spherical aberration, and as the exact position of the irradiated nanoparticle within the focal region is unknown, the absorption is basically impossible to calculate theoretically.

We developed a lipid based assay to directly measure this heating. The assay requires no pre-knowledge about the system apart from the phase-transition temperature of the bilayer. The temperature increase of an irradiated metallic nanoparticle can easily exceed hundreds of degrees Celsius.

Heating of elongated structures, as gold nanorods, is highly dependent on their orientation with respect to the laser polarization, as is evidenced in the image to the right.


Membrane tethers

In nature, cells make use of membrane-based tethers for a variety of purposes - for instance to adhere to a surface or to communicate with other cells.

Pulling a membrane tether for investigations of curvature adherence of surface proteins.

Using optical traps, we extract membrane tethers and investigate their force-extension properties. The model experiments are paralleled with experiments on tethers pulled from living cells. Also, we are interested in how particular proteins prefer to partition accordingly the the different curvatures present in this model system.

Weak interaction potentials

Using optical manipulation of lipid vesicles or microscopic/nanoscopic particles we probe typical weak interactions present in biological systems. For instance between a vesicle and a flat bilayer and how this changes upon insertion of proteins. In comparison to assays involving living cells, this model system has the advantage that the contribution from the individual constituents, e.g. a protein, can be isolated and monitored individually.

To simplify the picture, extract the most important constituents of the system. Membrane tethers, curvature sensitive proteins.