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

Technical Specifications

A particle image velocimetry system has been constructed by combining an Olympus IX 71 microscope, a 20X, 0.4 NA objective, and a TSI microPIV system (TSI Inc., MN).  A 45° mirror has been coated with a double layer of Cr (50 nm) and Au (200 nm) using sputtering techniques and has been aligned to distribute side-view incident light from a mercury lamp or Nd-YAG 532 nm laser to an objective beneath the stage.   An alternative setup was used for side-view image capturing of cells, involving the use of a second mirror to redirect light from a condenser bulb above the stage according to the techniques of Cao and colleagues [41].  To ensure proper alignment, an aluminum chassis was fabricated that holds the channel and mirrors firmly in place.

Flow Chamber Design

Two open-ended rectangular glass microslides (Vitrocom, Mountain Lakes, NJ) were used to form a 700x550 μm² channel according to the methods of Cao and colleagues [41].   A smaller microslide, with outer dimensions 50 mm x 0.15 mm x 0.6 mm was inserted into a larger microslide with inner dimensions 50 mm x 0.7 mm x 0.7 mm.  Prior to use, the microslides were washed successively in acetone, 93% ethanol, and double distilled water.  Preceding these washes, the 93% ethanol and double distilled water were both perfused through 0.20 µm polycarbonate filters to eliminate impurities.  Flow was controlled by means of a syringe pump.

Particle Image Velocimetry (PIV)

A TSI microPIV system (TSI Inc., MN) has combined with an Olympus IX 71 microscope and a 20X, 0.4 NA objective.  This system was used to obtain side-view PIV data in the form of double frame pairs using a Nd-YAG 532 nm laser light source.  The INSIGHT 6.0 software package was used to analyze the data (TSI Inc., MN).

1 μm-diameter orange fluorescent polystyrene microspheres of density 1.05 g/cm³ (Molecular Probes, Inc., OR) were used as tracer particles at a concentration of 0.0175% by volume.  For flow experiments involving cells, the microspheres were suspended at a concentration of 0.0524% by volume in RPMI-1640 media lacking phenol red (Gibco Laboratoies, Long Island, NY).  To reduce the non-specific binding of the spheres to each other and to the endothelial monolayer, this solution was supplemented with 5% bovine serum albumin (Sigma-Aldrich, Inc., St. Louis, MO) and incubated for 24 hrs at 37°C.  Prior to experimentation, the solutions were cooled to room temperature and further diluted in RPMI-1640 media to reach the desired microsphere concentration of 0.0175% by volume.  The viscosity of this solution was measured with a RotoViscoI cone-plate viscometer (ThermoMC, Madison, WI) to be approximately 1.5 cP at room temperature (18.5°C).

Background Processing

The background processing feature available within the INSIGHT 6.0 software package was very useful in reducing noise resulting from the illumination of tracer particles outside the focal plane.  For a given set of frame pairs, each consisting of a first capture “A” and a second capture “B”, the average intensities at each individual pixel were computed separately for the set of Frame A’s and for the set of  Frame B’s.  Subsequently, these values were subtracted from every Frame A and every Frame B respectively.

(a)     (b)

(a) An individual frame prior to background processing. (b) The same frame following background processing.

Image Stacking

The volume illumination element of microscale PIV dramatically increased background noise levels, necessitating the use of very low concentrations of microsphere tracer particles.  To increase the apparent density of tracer particles, images were stacked with ImageJ software (National Institutes of Health, Bethesda, MD) following background processing.  For example, to create a single 10X frame pair from 10 original frame pairs, all of the first captures (Frame A’s) within each of the original pairs would be added together and all of the second captures (Frame B’s) within each of the original pairs would be added together.  For experimental analysis, sets of 25 to 40 10X or 20X frame pairs were created.

 (a)    (b)  

 (c)   (d)

(a),(b) Two captures following background processing (c) An image created by stacking the captures in (a) and (b). (d) A 10X stack made from the same original set of frame pairs as (a) and (b).

Ensemble PIV Processing

Following image stacking, data was imported into INSIGHT 6.0 for analysis.  The image field was grided into a set of interrogation windows with a 50% overlap to satisfy Nyquist sampling and the data was processed using a direct correlator or FFT correlator.  Since only cases of steady state flow were considered, in which the velocity profile did not change during the time when acquiring data, the signal to noise ratio was greatly improved by ensemble averaging the correlation function across all frame pairs according to the techniques outlined by Meinhart and colleagues [46].

Endothelial Cells

Fibroblast L-cells that had been transfected to express human ICAM-1 (EI cells; provided by Sr. S. Simon, UC Davis) were maintained in culture as described by Gopalan and colleagues [47].

Leukocytes

T-leukemic Jurkat cells (provided by Dr. M. Lawrence, University of Virginia, Charlottesville, VA) were maintained in a humidified 5% CO₂ tissue culture incubator at 37°C.  They were cultured in a suspension of RPMI 1640 medium supplemented with 10% FBS, L-glutamine (0.292 mg/ml), penicillin (100U/ml) and streptomycin sulfate (100 µg/ml) (Gibco Laboratoies, Long Island, NY).  Prior to use in flow experiments, the Jurkat cells were incubated with phorbol myristate acetate (PMA) at a concentration of 200 ng/ml for 20 minutes at 37°C to promote LFA-1-dependent cell adhesion.

Theoretical Analysis

The pertinent equations for a flow assay with the side-view flow chamber have been derived by Cao et. al. [41].  The shear rate  under a given volumetric flow rate Q is given by

where W and H are the width and height of the flow channel respectively and F is the correction factor for a 3-dimensional flow profile.

            Within the central third of the flow region of the 700x550 μm² channel (W/H = 1.27), the mean F value = 1.45.  All quantitative flow data was acquired from focal plans within the central third of the flow region, where the actual wall shear rates do not deviate by greater than 3.6 % from the shear rate calculated using F = 1.45.