Direct 2D measurement of time-averaged forces and pressure amplitudes in acoustophoretic devices using optical trapping

Ultrasonic standing waves are increasingly applied in the manipulation and sorting of micrometer-sized particles in microfluidic cells. To optimize the performance of such devices, it is essential to know the exact forces that the particles experience in the acoustic wave. Although much progress has been made via analytical and numerical modeling, the reliability of these methods relies strongly on the assumptions used, e.g. the boundary conditions. Here, we have combined an acoustic flow cell with an optical laser trap to directly measure the force on a single spherical particle in two dimensions. While performing ultrasonic frequency scans, we measured the time-averaged forces on single particles that were moved with the laser trap through the microfluidic cell. The cell including piezoelectric transducers was modeled with finite element methods. We found that the experimentally obtained forces and the derived pressure fields confirm the predictions from theory and modeling. This novel approach can now be readily expanded to other particle, chamber, and fluid regimes and opens up the possibility of studying the effects of the presence of boundaries, acoustic streaming, and non-linear fluids.ISSN:1473-0197ISSN:1473-018

Propranolol restricts the mobility of single EGF-receptors on the cell surface before their internalization

The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process

Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm(−1)) than a viral protein-capsid coat and sustains deformations of one-third of the virion's diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host

Cell Visco-Elasticity Measured with AFM and Optical Trapping at Sub-Micrometer Deformations

The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components

Propranol induces stalling of EGFR.

<div><p>a) Trajectory of an EGFR after propranolol treatment. After some time the receptor got stuck and remained at one location (indicated in blue).</p> <p>b) The same trajectory plotted against time. The stationary part (blue) is clearly distinguishable. After being stationary for almost 40 s the receptor continued its travel again. </p></div

Tracking single EGFR.

<div><p>a) The bright-field and b) fluorescence image of two cells. On the cells typically multiple mobile QDs were visible. The coverslip is covered with immobilized QDs. </p> <p>c, d) Two typical trajectories of single EGFR recorded over one minute. </p> <p>e, f) The corresponding SD/CSD-plots. The SD steps between successive frames are shown in grey and the cumulative plot in green. In some curves apparent changes in slope could be observed (c), but the majority of CSD plots looked like (d) without obvious variations in slope.</p></div