In vivo multispectral, multiparameter, photoacoustic lymph flow cytometry with natural cell focusing, label-free detection and multicolor nanoparticle probes

Abstract

Compared with blood tests, cell assessment in lymphatics is not well-established. The goal of this work was to develop in vivo lymph tests using the principles of flow cytometry. Cells in living animals were counted by laser (420-2,300 nm) generation of photoacoustic (PA) signals in individual cells hydrodynamically focused by lymph valves into a single file flow, and using endogenous absorption as intrinsic cell-specific markers, or gold nanorods, nanoshells, and carbon nanotubes as multicolor probes. PA data were verified by high-speed transmission, photothermal, and fluorescent imaging. Counting of melanoma and immune-related cells in normal, apoptotic, and necrotic states in lymphatics in vivo was demonstrated to have the unprecedented sensitivity as one metastatic cell among millions of white blood cells. The time-resolved PA spectral identification of flowing cells was achieved using multicolor labels and laser pulses of different wavelengths and time delays. Multiparameter, noninvasive, portable flow cytometer can be used for preclinical studies on animals with the potential of translation to humans for in vivo PA mapping of colorless lymph vessels and sentinel nodes with simultaneous single cell detection and metastasis assessment without labeling or use of contrast dyes and/or novel low-toxic multicolor probes with different absorption spectra.

Figure 1

Schematic of in vivo flow cytometer integrating PA, PT, fluorescence, and TDM detection techniques.

Figure 2

(a) TDM image of lymph and blood microvessels of rat mesentery (10× magnification). (b) Fluorescent image of blood (green) and lymph (red) microvessels in mouse ear stained in vivo. (c) TDM image of a mouse ear fragment : A, arteriole; V, vein; L, EB-stained lymph vessel; linear (red) and the circular (green) laser beams on a lymph vessel. (d,e) Fluorescent images of a mesenteric lymphatic after intravenous injection of FITC and ICG, respectively. (f) Photographs of a melanoma tumor in mouse ear on the 15^th day after tumor inoculation. (g) Small melanoma cell clusters in the lung (tissue section) on the 30 ^th day after tumor inoculation.

Figure 3

(a) Principle of lymph test in vivo. (b) Dynamics of cell motion in the lymphatics. P₁ and P₂ is the pressure in the distal and in the proximal lymphangion, respectively. (c) High-speed TDM imaging of cells after a valve at 10× (top) and 100× (bottom) magnifications. (d) TDM images of cells in the central portion of a lymphangion in diastole (top) and systole (bottom). (e) Lymph vessel before (top) and after (bottom) one laser shot (wavelength, 585 nm; pulse width, 8 ns; beam diameter 100 μm, fluence 0.5 J/cm² ).

Figure 4

Transmission (magnification, 100×), fluorescence (magnification, 100×), and PT images (magnification of 20×) , together with integrated PT thermolens signals from a single rat lymphocyte, a single rat RBC, and a single B16F10 mouse melanoma cell in vitro. For fluorescence imaging, cells were stained with FITC. Parameters for PT/PA detection were as follows (amplitude, time scale, laser wavelength, and fluence): lymphocytes, 10 mV/div, 4 μs/div, 550 nm, and 100 mJ/cm²; RBC and melanoma cells, 20 mV/div, 4 μs/div, 580 nm, and 100 mJ/cm². Laser pump beam diameter was 25 μm.

Figure 5

PA signals, real (top) and compressed with the positive part only (bottom) from (a) melanoma cells in an ear lymphatic in an ear tumor model at second week after inoculation, and (b) a single RBC among lymphocytes. PA signal from (c) necrotic lymphocytes labeled with GNRs absorbing at 639 nm, (d) apoptotic lymphocytes labeled with GNSs absorbing at 865 nm, and (f) live neutrophils labeled with CNTs absorbing at both wavelengths . Laser parameters (wavelength, fluence) (a) 850 nm, 30 mJ/cm²; (b) 530 nm, 100 mJ/cm²; (c-e) 639 nm and 865 nm, 25 mJ/cm² and 30 mJ/cm², respectively.

Figure 6

Image lymph flow cytometry. (a) The relationships between initial lymphatics, afferent lymphatics, lymph nodes, and efferent lymphatics. (b,c) Cells passing through the endothelial wall of an initial lymphatic (magnifications, 4× and 100×, respectively). (d-h) High-resolution high-speed TDM images of a WBC, a RBC, a microparticle (likely chylomicron), a melanoma cell aggregate containing two cells and a partly lysed single melanoma cell in lymph flow (magnification, 40×; 500-2,500 fps), respectively. (i) B-lymphocyte exiting a valve aperture. (j) Fluorescent image of an apoptotic cell in the lymphatic lumen (dash lines). (k,l) Images of a mesenteric lymph node fragment in vivo at different magnifications (4× and 100×, respectively)