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 ~100 pN/s, 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.
Digital holographic interferometry in combination with a flexible fiber endoscope allows high precision measurements of deformations on hidden objects surfaces, inside cavities and objects with small access apertures. A digital holographic endoscopy system is described with a frequency-doubled, twin oscillator Q-switched pulsed Nd:YAG laser as light source. A sequence of digital hologram pairs are recorded with a maximum repetition rate of 260 ms. Each digital hologram is captured at separate video frames of a CCD-camera. The time separation between the laser pulses from each cavity can be set in the range from 50 to 500 μs. The digital holograms are transferred to a PC via a frame grabber and evaluated quantitatively by the Fourier transform method. The resulting phase fringe pattern has the information needed to evaluate quantitatively the amount of the deformation. Experimental results of vibration measurements of hidden mechanical and biological object surfaces are presented. The quality of the results obtained by mechanical object surfaces is usually higher than for biological surfaces. This can be explained easily by the fact that a biological surface is much more complex than a mechanical surface in the sense that some parts of the surface may reflect the light well whereas other parts may absorb the light. Also, biological surfaces are translucent, which means that part of the light may enter inside the sample where it may be absorbed or reflected.
The ability of uropathogenic Escherichia coli (UPEC) to cause urinary tract infections is dependent on their ability to colonize the uroepithelium. Infecting bacteria ascend the urethra to the bladder and then kidneys by attaching to the uroepithelial cells via the differential expression of adhesins. P pili are associated with pyelonephritis, the more severe infection of the kidneys. In order to find means to treat pyelonephritis, it is therefore of interest to investigate the properties P pili. The mechanical behavior of individual P pili of uropathogenic Escherichia coli has recently been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of ~1000 PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. We have earlier studied P pili regarding its stretching/elongation properties where we have found and characterized three different elongation regions, of which one constitute an unfolding of the quaternary (helical) structure of the PapA rod. It was shown that this unfolding takes place at an elongation independent force of 27 ± 2 pN. We have also recently performed studies on its folding properties and shown that the unfolding/folding of the PapA rod is completely reversible. Here we present a study of the dynamical properties of the PapA rod. We show, among other things, that the unfolding force increases and that the folding force decreases with the speed of unfolding and folding respectively. Moreover, the PapA rod can be folded-unfolded a significant number of times without loosing its characteristics, a phenomenon that is believed to be important for the bacterium to keep close contact to the host tissue and consequently helps the bacterium to colonize the host tissue.
Optical tweezers together with a position sensitive detection system allows measurements of forces in the pN range between micro-sized biological objects. A prototype force measurement system has been constructed around in inverted microscope with an argon-ion pumped Ti:sapphire laser as light source for optical trapping. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omni-directional mechanical spring if an external force displaces it. The displacement from the equilibrium position is a measure of the exerted force. For position detection of the trapped particle (polystyrene beads), a He-Ne laser beam is focused a small distance below the trapping focus. An image of the bead appears as a distinct spot in the far field, monitored by a photosensitive detector. The position data is converted to a force measurement by a calibration procedure. The system has been used for measuring the binding forces between E-coli bacterial adhesin and their receptor sugars.
A system based on digital holographic interferometry in combination with an endoscope is described. A Q-switched pulsed laser is used. Two digital holograms of the test object, corresponding to the two laser pulse, are captured at separate video frames of the CCD-camera, transferred in a frame grabber and further processed in a PC. If during the interval between the two laser pulses the object undergoes a deformation, a fringe pattern will result from the difference between the two holograms. This fringe pattern has the information needed to quantitatively evaluate the amount of the deformation. A compact system has been developed to be used for many applications, both mechanical and biological, where measurements need to be performed at 'hidden' surfaces or inside more or less closed objects. The quality of the result obtained by using mechanical objects is usually better, this can be easily explained by the fact that a biological surface is much more complex, in particular some parts reflect well the light and some other absorb it. A comparison of different endoscopes as object image carrier in pulsed digital holography is presented.
Image plane digital holography is used to evaluate the 3D vector components and object contour of a dynamically moving object. Pulses from a Q-switched ruby laser are used to record image plane holograms on the faceplate of a CCD camera, to be later digitally reconstructed. Three different illumination directions are used to get displacement information along three different sensitivity vectors. The latter are combined into a 3D resultant displacement vector for every object point. To compete the full 3D analysis the object contour is measured with the two-wavelength method. Data for contour and 3D-displacement are combined and displayed.
An optoelectronic system is presented that is used to measure the 3D vector components of a vibrating object. The complete determination of the vibration occurs by combination of three different measurements. (1) A 3D system based on digital holography for the measurement of the 3D deformation vector along the directions x, y and z is used. Pulses from a ruby laser, with a separation in the range from 1 to 1000 microseconds, are used to record holograms on CCD sensors, which are later digitally reconstructed. (2) The shape of the object is determined. In this case too the ruby laser is used as a light source but now two pulses are emitted with different wavelengths. The wavelength change is produced by changing the distance between the plates of the laser output etalon. The shape is obtained by subtraction of the phases of the wavefronts recorded at the two different wavelengths. (3) For the absolute measurement of a vibration using double exposure holographic techniques, it is necessary to have a reference point where the absolute vibration is known. A precise measurement of the vibrations in a point is done by using a 3D laser Doppler vibrometer.
An optoelectronic system based on digital holography is used to measure the three dimensional vector components and object shape of a vibrating object. Pulses from a ruby laser, with a separation in the range from 1 to 1000 microseconds, are used to record holograms on CCD sensors, which are later digitally reconstructed. Three different illumination directions are used to get the deformation along three different sensitivity vectors, that are afterwards combined into a 3D resultant deformation. To measure the shape of the object the two- wavelength method is used. The wavelength change is produced by changing the distance between the plates of the laser output etalon, thus obtaining the shape by subtracting the phases of the wavefronts recorded at those wavelengths. The data sets for the shape and 3D-deformation are combined and graphically shown. Finally, by using a non linear crystal (BBO) it was possible to double the frequency of the radiation emitted by the ruby laser allowing to get two wavelengths (694 nm and 347 nm) simultaneously and thus to record digital holograms with different sensitivities.
The density distribution in the air surrounding an airgun muzzle is visualized using pulsed TV holography. A ruby laser emitting short coherent light pulses (30 ns) is used as light source. Pulsed holograms are captured by a CCD-camera and their optical phase difference is evaluated by means of the Fourier transform method. A number of experiments are performed where the event is recorded at different instants of time as the lead bullet is about to leave the muzzle. Phase maps showing the integrated density distribution are presented. A jet with decreasing density ahead of the bullet can be observed. At the moment the bullet leaves the muzzle, a spherical sound pressure wave starts to propagate out into the air. The density in the jet in front of the traveling bullet can be calculated by means of full shock-wave theory. The theory confirms the decrease in density observed in the measurements.
An all electronic double-pulsed TV holography system is described. Two separate image-plane holograms are recorded using a CCD camera. A small angular offset between the reference and object beams introduces carrier fringes in the image plane. Thus, the Fourier spectrum of the image contains distinct parts that can be filtered out and inverse transformed to yield the phase information. Experiments showing propagating transient bending waves in an aluminum plate generated by a focused laser pulse are performed. Phase maps of good quality are unwrapped and presented as 3D plots of the out-of-plane displacement. The system has also been used to evaluate the 3D distribution of transient acoustic fields in air generated by an electrical discharge. Several projections of the acoustic field is recorded and the 3D pressure field is reconstructed by computerized tomography.
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