In vivo acoustic and photoacoustic focusing of circulating cells

Abstract

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models.

Figure 1. Principle of cell manipulation in vivo.

(a) Schematic of in vivo flow cytometry with acoustic focusing and PA detection of circulating cells and nanoparticles. (b) Nude mouse ear-vessel model. (c) Cross-section of an acoustic resonator around a selected vessel in mouse ear skin. (d) Principle of PA focusing of flowing cells with two linear laser beams creating “virtual PA walls”. (e) Cell redirection between two blood vessels with a linear laser beam creating a virtual PA wall.

Figure 2. In vitro acoustic focusing of blood cells in flow.

(a) Schematic of acoustic focusing (left) and experimental setup using a quartz capillary with a 100-μm inner diameter (right). (b) Distribution of mouse RBCs in flow before (left) and during (right) the application of ultrasound Ultrasound parameters: frequency, 7.29 MHz; intensity 0.5 W/cm². (c) Schematic of the acoustic focusing of blood flow in the gap between two capillaries. (d) Leakage of blood flow between two capillaries (left) and acoustic canalization of blood flow in the gap between two capillaries (right). Ultrasound parameters: frequency, 0.6 MHz; intensity, 1.8 W/cm².

Figure 3. PA manipulation of beads and cancer cells in vitro.

Polystyrene beads (a–d,f–h) and melanoma cells (e) were manipulated in vitro in a glass capillary filled with ICG in water (a–d: 6.8–μm beads) and in mouse blood (f–h): 25–μm beads with the use of linear laser beams of different spatial configurations. Laser parameters: wavelength, 820 nm (a–c,f–h); 671 nm (bottom) and 820 nm (top) (e); 671 nm (first) and 820 nm (second) (d); pulse width, 8 ns; pulse rate, 10 kHz; distance between two beams 20 μm (d); pulse energy, 5 μJ (energy fluence, 0.02–0.1 J/cm²).

Figure 4. In vivo cell focusing in blood and lymph flow in living animals using acoustic waves.

(a) Blood flow before (left) and after (right) application of ultrasound in a mouse ear vessel. The dashed lines mark the vessel’s boundary. (b) Asymmetric displacement of blood flow to a vessel wall under ultrasound action (right), compared to control (left). (c) Acoustic stopping of blood flow in a localized zone (right), compared to control (left). (d,e) Acoustic focusing of cells (WBCs) in an 180-μm-diameter mouse mesenteric lymph vessel under the influence of acoustic standing waves in two different vessels. Ultrasound parameters: frequency, ~3 MHz; intensity, 0.4 W/cm² (d) and 1 W/cm² (e). Average flow velocities: 5 mm/s (blood); 1.5 mm/s (lymph).

Figure 5. PA manipulation of cells in vivo.

(a) Blood flow before (left) and after (right) laser irradiation in the area of mouse ear vessels indicated by the arrow. (b) Cells in lymph flow of a mouse mesentery vessel before (left) and after (right) irradiation with a linear laser beam. The passage of the laser beam through vessels blurred it into an ellipsoidal shape as seen in image due to light scattering in the tissue. (c) Trapping of a single cell in a mouse mesenteric lymph vessel with a ring-shaped laser beam (right). Laser parameters: wavelength, 532 nm (a) and 820 nm (b,c); energy fluence, 0.7 J/cm² (a) and 60 mJ/cm² (b,c).

Figure 6. In vivo PAFC with acoustic cell focusing.

(a) In vivo label-free PA detection of individual RBCs in focused flow (right) compared to nonfocused flow (left) using a planar acoustic resonator. (b) Optical (transmission) images of normal (left) and sickle (right) RBCs. (c) In vivo time-resolved PA detection of individual WBCs (leukocytes) molecularly targeted by gold nanorods (GNRs) conjugated with antibody to CD45 receptors in focused blood flow in mouse ear microvessels (right) compared to the overlapping peaks from WBCs in nonfocused flow (left). Laser parameters: wavelength/fluence, 532 nm/40 mJ/cm² (a) and 820 nm/50 mJ/cm² (c). Ultrasound parameters: frequency, 3 MHz; intensity, 0.5 W/cm².