Design of Side-View Particle Image Velocimetry System for Cellular Adhesion Analysis

To demonstrate the capability of the side-view imaging system, top-view and side-view image were captured after attaching 16 µm-diameter green fluorescent polystyrene divinylbenzene (DVB) microspheres (Duke Scientific Corp., CA) 16 µm-diameter fluorescent beads onto the bottom wall of the 700x550 μm² channel.  Shown below are the top and side-views of two beads visualized using a mercury light source.  The beads are easily distinguished by changing the focal plane in the z-direction.

Velocity Profile of Wall Shear Rate within Microcapillary

In order to validate the approach of side-view PIV in acquiring accurate flow data, flow was perfused through the 700x550 μm² channel at a rate of 73 μl/min.  Using the side-view PIV techniques outlined Chapter 2, the velocity vectors near the bottom wall were calculated.  Their values show a well matched correspondence to the theoretical velocity values near the wall.

Acquire Velocity Profile over Simulated Adherent Cell

A 16 µm-diameter fluorescent bead was attached to the bottom wall of the 700x550 μm² channel and flow was subsequently perfused at a rate of 73 μl/min with a syringe pump.  The tracer particles were illuminated with a Nd-YAG 532 nm laser with a 600 µsec delay between pulses the side-view PIV techniques described in Chapter 2 were used to calculate a velocity vector field.

Velocity Profile over Leukocyte

A leukocyte suspension was perfused within the 700x550 μm² channel.  For a period of approximately 5-10 min, flow was suspended and the cells were permitted to adhere before flow was reapplied at 73 µl/min.  A rather large cell (estimated at 24.5 µm in width and 20.7 µm in height) was chosen so as to give a maximum disruption in the flow field.  The tracer particles were illuminated with a Nd-YAG 532 nm laser with a 600 µsec delay between pulses the side-view PIV techniques described in Chapter 2 were used to calculate a velocity vector field.  The interrogation windows were 30 x 20 pixels in size (width x height) and pixel size is estimated to be 0.225 µm/pixel.

Shear Rate Analysis

A large Jurkat cell of approximately 22 µm in diameter was adhered under static conditions to an EI monolayer within the 700x550 μm² channel.  Flow was applied at 73 ul/min and 365 ul/min to correspond to low and high shear cases of 50 s^-1 and 250 s^-1 respectively.  The tracer particles were illuminated with an Nd-YAG 532 nm laser with 600 and 150 µsec time delays between pulses for the low and high shear cases respectively.  The side-view PIV techniques described in Chapter 2 were used to calculate a velocity vector field (Figure 3.10).  The interrogation windows were 30 x 20 pixels in size (width x height).

The upstream wall shear rates values were verified (Figure 3.13) by plotting the x-directed velocity values from three columns of vectors calculated using interrogation windows of 128 pixels in width and 12 pixels in height where the pixel size is approximately 0.225 µm/pixel.  Linear regressions were calculated from data points less than 55 µm above the channel wall (10% of channel height) using Excel software (Microsoft Corporation, Redmond, WA) where the intercept of the x-directed velocity was assigned to be at the location of the wall.  The slopes of the regression lines, corresponding to 51.0 and 257.7, are taken to be the numerical values of the upstream wall shear rates in the 73 ul/min (low shear) and 365 ul/min (high shear) cases respectively.  These wall shear rates correspond to wall shear stresses of 0.765 dyne/cm² and 3.864 dyne/cm².  In order to be sure that deviations from a linear flow profile near the wall were not altering the calculated upstream wall shear rates, quadratic regression curves were also calculated using x-directed velocity values from points up to 170 µm above the channel wall (30.9% of channel height).  As in the linear case, the intercepts were assigned to be at the channel wall location (endothelial cell layer).   The values of the slopes at the location of the wall were within 10% of the corresponding values calculated from the linear regressions.

Significant deformation characterizes the shape of the cell under both low and high shear when compared to static conditions.  For analyzing the pertinent shear rates over the surface of the cell in each case, velocity values were calculated over the surface of the cell using side-view PIV techniques utilizing interrogation windows approximately 14.4 µm in width and 2.7 µm in height (see Figure 3.11).  The values of the velocity components in the direction of flow were plotted (see Figure 3.12) and quadratic regression curves were fitted.  The slopes of the curves at the point of zero x-directed velocity were taken to be the shear rates above the cell.  For comparison purposes, as well as for validation of the flow perfusion rates, the upstream wall shear rates were calculated (see Figure 3.13) using side-view PIV techniques to acquire velocity vectors near the EI monolayer and measuring the slope of the calculated linear regression curve.

The values of the shear rates above the cell were found to be 103.4 s^-1 and 417.9 s¹ and low and high shear cases respectively.  These yielded values of 2.03 and 1.62 for the ratio of shear rate above the cell to upstream wall shear rate for the low and high shear cases.  Thus, under the high deformation of the high shear case, the ratio of the shear forces acting on the cell surface to those acting at the upstream wall is 20.2% lower.