Multinode acoustic focusing for parallel flow cytometry

Abstract

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 μm, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multinode acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 μm in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of a CD4+ cellular immunophenotyping assay. This approach will have significant impact toward the creation of high throughput flow cytometers for rare cell detection applications (e.g., circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.

Figure 1

Multinode acoustic focusing in glass devices. (a) A schematic drawing of acoustic flow cell made with a rectangular glass capillary. (b) The location of first three pressure nodes for a fixed width capillary (Top – width = λ/2, center – width = λ, bottom – width = 3/2 λ). Optical micrographs showing the focusing of two sizes of polystyrene particles into 3 nodes: (c) 10 μm in a 0.1 × 1 mm capillary and (d) 107 μm in a 0.2 × 2 mm capillary.

Figure 2

Analysis of multinode acoustic focusing in 0.1 (height) × 1 (width) mm glass capillaries. Fluorescent micrographs of (a) non-focused and (b) focused streams of 10 μm NR-ps particles. (c) Fluorescent image analysis of non-focused and (d) focused streams. Flow cytometric analysis of (e) non-focused and (f) focused streams.

Figure 3

Analysis of WBC in multinode acoustic flow cell. (a) Contour plot of stained WBC (b) Histogram of gated lymphocytes population with 40 % of CD4+ cells.

Figure 4

Flow cytometric analysis of acoustic focusing of 107 μm red fluorescent polystyrene particles flowing at 1.6 mL/min. Each of the 3 focused streamlines was analyzed individually yielding CVs for the left (a) 5%, center (b) 6%, and right (c) 8% streams respectively.

Figure 5

Formation of highly parallel focused streams in the machined device with different resonance frequencies. (a) A schematic of the machined flow cell for high numbers of parallel streams. The left hand photo panels show focusing captured at long range, while the right hand panels show the streams at close range. (b, d) 1.17 MHz, 24 streams of 10 μm polystyrene particles, (c, e) 1.54 MHz, 33 streams of 10 μm polystyrene particles. (f, g) 1.54 MHz, 33 streams of red blood cells.

Figure 6

Observed and theoretically expected number of focused streams of 10 μm NR-ps particles generated at varying applied resonance frequencies (circles – observed, line – expected)