Validation, In-Depth Analysis, and Modification of the Micropipette Aspiration Technique

Abstract

The micropipette aspiration technique (MAT) has been successfully applied to many studies in cell adhesion such as leukocyte-endothelium interactions. However, this technique has never been validated experimentally and it has been only employed to impose constant forces. In this study, we validated the force measurement of the MAT with the optical trap and analyzed two technical issues of the MAT, force-transducer offset and cell-micropipette gap, with finite element simulation. We also modified the MAT so that increasing or decreasing forces can be applied. With the modified MAT, we studied tether extraction from endothelial cells by pulling single tethers at increasing velocities and constant force loading rates. Before the onset of tether extraction, an apparently-linear surface protrusion of a few hundred nanometers was observed, which is likely related to membrane receptors pulling on the underlying cytoskeleton. The strength of the modified MAT lies in its capability and consistency to apply a wide range of force loading rates from several piconewtons per second up to thousands of piconewtons per second. With this modification, the MAT becomes more versatile in the study of single molecule and single cell biophysics.

FIGURE 1

(a) One video micrograph showing tether extraction from a human neutrophil using the OT and the MAT simultaneously. An antibody-coated bead and a passive human neutrophil were used as the force transducers of the OT and the MAT, respectively. (b and c) Two video micrographs showing tether extraction from a suspended (b) or attached (c) HUVEC with an antibody-coated bead (the force transducer of the MAT). A voltage stamp of 5V (shown actually as “+05.08”) was used to indicate the instant when the stage started to move and the suction pressure started to increase.

FIGURE 2

Geometric dimensions used in the finite element simulations. (a) Model I: L_p = 60 μm, R_p = 4 μm, R_b = 3.9 μm, and D_pb = 0–0.08 μm. (b) Model II: H = 30 μm, L_f = 40 μm, R_p = 4 μm, R_b = 3.9 μm, T_p = 3 μm, D_bp = 8 μm, H_c = 4 μm, and L_c = 20 μm. The total pipette length was 650 μm and only part of the pipette is shown. For this particular case, the distance between the cell apex and the pipette tip (D_pc) was 2 μm. The size of the entire simulation area outside the pipette was 30 μm × 40 μm.

FIGURE 3

(a) Pulling forces (F) measured simultaneously with the MAT and the OT during tether extraction from human neutrophils. Although the force magnitudes distributed widely from 40 pN up to 100 pN due to the cell heterogeneity, the close agreement between the MAT and OT is clear. The stiffness of the OT was ~0.08 pN/nm and the suction pressure in the micropipette was 2 pN/μm². (b) Correlation between the pulling forces measured simultaneously with the OT and the MAT.

FIGURE 4

Effect of D_pb on the relative error (E_r) between the two forces calculated from either Eq. 1 (F_Eq) or FIDAP simulation (F_FEM). F_FEM is also shown. Five different D_pb values in the range of 0 to 0.08 μm were examined at the constant total pressure drop of 2.5 pN/μm² and the tether growth velocity of 10 μm/s.

FIGURE 5

Effect of D_pc on the inner pressure drop in the MAT (a) and the force exerted on the transducer, F (b). Under the constant total pressure drop of 4.24 pN/μm², six different distances (D_pc) were simulated with the bead velocity at either 7.5 μm/s or 10 μm/s, simulating tether extraction at two different velocities.

FIGURE 6

(a) A typical case of the bead free motion obtained with the modified MAT at the stage speed of 75 μm/s. A quadratic equation was used to fit the bead displacement and the resulting correlation coefficient is also shown in the figure. (b) A typical case of the bead displacement acquired at the stage speed of 75 μm/s when there was adhesion between the bead and a suspended HUVEC. Both (a) and (b) were acquired with the same pair of bead and micropipette. Two different regimes, surface protrusion and tether extraction, were identified from the bead displacement. The termination of the initial surface protrusion could be clearly identified in the inset figure, which also indicates the onset of tether extraction.

FIGURE 7

Further analysis of surface protrusion and tether extraction of the case shown in Fig. 6b. A quadratic equation was fitted very well to the bead displacement in the tether extraction regime (D = 0.80t² − 6.12t +18.30), so a linear relationship was found between the pulling force and the tether growth velocity (a). In the surface protrusion regime, the cell surface deformed linearly with a protrusional stiffness of 320 pN/μm (b) and a constant force loading rate of 34 pN/s (data not shown).

FIGURE 8

Effect of the force loading rate on surface protrusion from HUVECs. Anti-CD31-coated beads were used as the force transducer to interact with both suspended and attached HUVECs. The corresponding protrusional stiffness (a) and crossover force (b) are all plotted as a function of the force loading rate. Each point here represents the mean value of 5~22 adhesion events obtained from three to ten cells. The error bars in (a) stand for the standard deviations in both directions.