Introduction

Conventional Eulerian velocimetry techniques provide a detailed assessment of the velocity field at a particular point in space and moment in time. This is achieved by measuring the spatial displacement of a large number of particles (1) obtained in two separate images at a known time interval. In many applications, these techniques can provide a detailed snapshot description of the flow field.

Blood flow, particularly in inflammation, can present a challenge for Eulerian approaches. Significant variability in local cell velocities has been observed in inflammation (2,3,4,5). Similarly, the cellular composition of the blood—including red cells and leukocytes—can create blood flow that is nonuniform. The nonuniformity of inflammatory blood flow is illustrated by leukocyte trajectories that involve margination mural interactions and prolonged residence times.

To provide a description of unsteady and nonuniform flow fields, Lagrangian methods (6) track the movement of individual particles (7). An advantage of Lagrangian descriptions is that these methods provide more information about the fate of individual particles and flow in the system. The disadvantage of a Lagrangian approach has been the technical demands of tracking individual particles in rapid biologic processes. The description of Lagrangian coordinates in a biologic system such as the microcirculation requires (i) intensely labeled particles (or cells) to enable detection within tissues; (ii) a detection system with sufficient temporal and spatial resolution to track particles at velocities up to 5 mm/s; and (iii) image analysis software that permits the accurate plotting of Lagrangian coordinates from video streams composed of thousands of acquired images.

We have previously described nanoparticles developed for defining flow fields in the microcirculation (7). In this report, we describe the application of recently developed electron multiplying charge-coupled device (EMCCD) cameras and image analysis software for the definition of Lagrangian coordinates in the microcirculation. In addition to improved tracking of individual particles, these techniques permit the simultaneous tracking of multicolored particles in vivo. The ability to define Lagrangian coordinates of different types of blood particles will provide a better understanding of the dynamics of both plasma and cell motion in the microcirculation.

Materials and Methods

Mice

Male Balb/c mice (Jackson Laboratory, Bar Harbor, ME, USA), with care consistent with guidelines of the American Association for Accreditation of Laboratory Animal Care (Bethesda, MD, USA), were used in all experiments.

Nanoparticles

The nanoparticles were developed by Molecular Probes (Invitrogen, Eugene, OR, USA) for intravascular particle tracking. These particles were of similar composition to those reported previously (8), but manufactured with superior fluorescent characteristics, smaller size, and low surface charge content (7). The surface charge of the nanoparticles used in this study ranged from 1.5 to 6.5 µEq/g. The nanoparticles were labeled with green (ex. 490 nm; em. 520 nm), orange (ex. 545 nm; em. 570 nm), and infrared (ex. 675 nm; em. 700 nm) fluorochromes. Although particles from 40 nm to 2 µm were investigated, 500 nm particles were used in most studies.

EMCCD Camera

Videomicroscopy recorded 14-bit fluorescent images using a C9100–02 EMCCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The C9100–02 has an air-cooled head and on-chip electron gain multiplication (2000×). Images with 1000 × 1000 pixel resolution were routinely obtained at 50 frames per second (fps). Frame rates exceeding 100 fps were obtained with binning and subarrays. The images were typically recorded in image stacks comprising 30-s to 10-min video sequences.

In Vitro Flow Chamber

The technical specifications of the flow chamber has been previously described (9,10). Briefly, the design features included 0.5 mm holes in both inlet and outlet manifolds to rapidly stabilize laminar flow and permit the use of standard microscope slides. Rounded fluid capacitors positioned at the ends of the flow deck dampened eddy currents at the higher flows. The flow chamber was perfused with a NE-1000 withdrawal syringe pump (New Era, Farmingdale, NY, USA). In most experiments, the perfusate was normal saline containing nanoparticles.

Intravital Fluorescence Labeling

A 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Molecular Probes, Invitrogen) labeling solution was prepared in dimethyl sulfoxide (DMSO) as described (11). The freshly prepared CFSE (400 µL) was injected into the tail vein of an anesthetized mouse over 2–3 min. The CFSE tracer (ex. 480 nm; em. 520 nm) was imaged with 25 nm band pass filters (Chroma Technology, Rockingham, VT, USA).