Erik Fällman

Design for fully steerable dual-trap optical tweezers

A design for complete beam steering (in three dimensions) of one or two optical tweezers traps is presented. The two most important requirements for efficient and stable movement of an optical trap are identified. A detailed recipe for the construction of a movable optical tweezers trap that fulfills these requirements is given (exemplified with an inverted microscope). The system has been found to allow for precise and free movements of both traps in all three dimensions in a dual-trap optical tweezers configuration and to be robust and reliable, as well as forgiving of small misalignments in the optical system.

Stress response in Caenorhabditis elegans caused by optical tweezers: wavelength, power, and time dependence.

Optical tweezers have emerged as a powerful technique for micromanipulation of living cells. Although the technique often has been claimed to be nonintrusive, evidence has appeared that this is not always the case. This work presents evidence that near-infrared continuous-wave laser light from optical tweezers can produce stress in Caenorhabditis elegans. A transgenic strain of C. elegans, carrying an integrated heat-shock-responsive reporter gene, has been exposed to laser light under a variety of illumination conditions. It was found that gene expression was most often induced by light of 760 nm, and least by 810 nm. The stress response increased with laser power and irradiation time. At 810 nm, significant gene expression could be observed at 360 mW of illumination, which is more than one order of magnitude above that normally used in optical tweezers. In the 700-760-nm range, the results show that the stress response is caused by photochemical processes, whereas at 810 nm, it mainly has a photothermal origin. These results give further evidence that the 700-760-nm wavelength region is unsuitable for optical tweezers and suggest that work at 810 nm at normal laser powers does not cause stress at the cellular level.

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic

P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of ∼103 PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.

Influence of a glass-water interface on the on-axis trapping of micrometer-sized spherical objects by optical tweezers

A systematic study of the influence of a glass-water interface on the on-axis trapping of micrometer-sized spherical objects by optical tweezers is presented. The ways in which the escape force and the trapping position, as well as the stiffness of the trap, depend on the focusing depth, the numerical aperture, and the degree of overfilling of the objective entrance pupil are investigated. It is concluded, among other things, that objectives with the highest numerical aperture and the use of large degrees of overfilling do not always provide the optimum trapping conditions at finite depths.

Physical Properties of Biopolymers Assessed by Optical Tweezers : Analysis of Folding and Refolding of Bacterial Pili

Bacterial adhesion to surfaces mediated by specific adhesion organelles that promote infections, as exemplified by the pili of uropathogenic E. coli, is studied mostly at the level of cell-cell interactions and thereby reflects the averaged behavior of multiple pili. The role of pilus rod structure has therefore only been estimated from the outcome of experiments involving large numbers of organelles at the same time. It has, however, lately become clear that the biomechanical behavior of the pilus shafts play an important, albeit hitherto rather unrecognized, role in the adhesion process. For example, it has been observed that shafts from two different strains, even though they are similar in structure, result in large differences in the ability of the bacteria to adhere to their host tissue. However, in order to identify all properties of pilus structures that are of importance in the adhesion process, the biomechanical properties of pili must be assessed at the single-molecule level. Due to the low range of forces of these structures, until recently it was not possible to obtain such information. However, with the development of force-measuring optical tweezers (FMOT) with force resolution in the low piconewton range, it has lately become possible to assess forces mediated by individual pili on single living bacteria in real time. FMOT allows for a more or less detailed mapping of the biomechanical properties of individual pilus shafts, in particular those that are associated with their elongation and contraction under stress. This Mi- nireview presents the FMOT technique, the biological model system, and results from assessment of the biomechanical properties of bacterial pili. The information retrieved is also compared with that obtained by atomic force microscopy.

Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod

The infectious ability of uropathogenic Escherichia coli relies on adhesive fibers, termed pili or fimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that are formed via a highly preserved assembly and secretion system called the chaperone-usher pathway. We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt both type 1 and P pilus biogenesis in E. coli. In this study, we show that the pilicides do not affect the structure, dynamics or function of the pilus rod. This was demonstrated by first suppressing the expression of P pili in E. coli by pilicide treatment and, next, measuring the biophysical properties of the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic force microscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers were determined by optical tweezers force measurements on individual pili and were found to be intact. The presented results establish a potential use of pilicides as chemical tools to study important biological processes e.g. adhesion, pilus biogenesis and the role of pili in infections and biofilm formation.

Unraveling the Secrets of Bacterial Adhesion Organelles Using Single-Molecule Force Spectroscopy

Many types of bacterium express micrometer-long attachment organelles (so called pili) whose role is to mediate adhesion to host tissue. Until recently, little was known about their function in the ...

Dual-trap technique for reduction of low-frequency noise in force measuring optical tweezers

High-resolution long-time force measurements by optical tweezers are often limited by low-frequency (1/f) noise. A dual-trap technique is presented that can reduce such noise in the force signal. It incorporates a second trap (a reference trap) that probes the noise in the system and it is based upon the assumption that the low-frequency parts of the noise from the two traps are correlated. A subtraction of the low-frequency signal from the reference trap from the signal from the force measuring trap will therefore yield a net signal that is significantly less influenced by noise. It is shown that this dual-trap technique can reduce the noise in the force signal up to 60% depending on detection bandwidth.

Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers

Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

Force measuring optical tweezers system for long time measurements of P pili stability

A force-measuring optical tweezers instrumentation and long time measurements of the elongation and retraction of bacterial fimbriae from Uropathogenic E. coli (UPEC) under strain are presented. The instrumentation is presented in some detail. Special emphasis is given to measures taken to reduce the influence of noise and drifts in the system and from the surrounding, which makes long term force measurements possible. Individual P pili from UPEC bacteria were used as a biological model system for repetitive unfolding and refolding cycles of bacterial fimbriae under equilibrium conditions. P pili have evolved into a three-dimensional helix-like structure, the PapA rod, that can be successively and significantly elongated and/or unfolded when exposed to external forces. The instrumentation is used for characterization of the force-vs.-elongation response of the PapA rod of individual P pili, with emphasis on the long time stability of the forced unfolding and refolding of the helical structure of the PapA rod. The results show that the PapA rod is capable of withstanding extensive strain, leading to a complete unfolding of the helical structure, repetitive times during the life cycle of a bacterium without any noticeable alteration of the mechanical properties of the P pili. This function is believed to be importance for UPEC bacteria in vivo since it provides a close contact to a host cell (which is an initial step of invasion) despite urine cleaning attempts.