In Vivo Multispectral Photoacoustic Lymph Flow Cytometry with Natural Cell Focusing and Multicolor Nanoparticle Probes

doi:10.1002/cyto.a.20587

Cytometry A. Author manuscript; available in PMC 2009 October 1.

Published in final edited form as:

Cytometry A. 2008 October; 73(10): 884–894.

doi: 10.1002/cyto.a.20587

PMCID: PMC2606693

NIHMSID: NIHMS79944

In Vivo Multispectral Photoacoustic Lymph Flow Cytometry with Natural Cell Focusing and Multicolor Nanoparticle Probes

Ekaterina I. Galanzha,^a^b Evgeny V. Shashkov,^a^c Valery V. Tuchin,^b and Vladimir P. Zharov^a^*

^aUniversity of Arkansas for Medical Sciences, Philips Classic Laser Laboratories, Little Rock, Arkansas 72205

^bSaratov State University, Institute of Optics and Biophotonics, Saratov 410012, Russia

^cProkhorov General Physics Institute, Moscow 119991, Russia

^*Correspondence to: Vladimir P. Zharov, PhD, Philips Classic Laser Laboratories, University of Arkansas for Medical Sciences, 4301 West Markham St. #543, Little Rock, AR 72205-7199, USA. Phone: 501-603-1213; fax: 501-686-8029 E-mail: ZharovVladimirP/at/uams.edu

The publisher's final edited version of this article is available free at Cytometry A

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Abstract

Background

Compared to 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.

Methods

Cells in living animals were counted by laser (420-2300 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 was verified by high-speed transmission, photothermal, and fluorescent imaging.

Results

Counting melanoma and immune-related cells in normal, apoptotic, and necrotic stages in lymphatics in vivo was demonstrated to have the unprecedented sensitivity as one metastatic cell among millions of normal cells. The time-resolved PA spectral identification of flowing cells was achieved using multicolor labels and laser pulses of different wavelengths and time delays.

Conclusions

Multispectral, non-invasive, portable flow cytometery 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.

Keywords: in vitro flow cytometry, lymphatics, sentinel lymph nodes, photoacoustic method, multicolor detection, nanoparticles, melanoma metastasis, immunity, apoptosis, necrosis

INTRODUCTION

Single-cell detection is important in many branches of biology and medicine, including the fundamental study of immune system function, natural cell death, and disease diagnosis. This detection is particularly important in assessing circulating tumor cells (CTCs) as markers of cancer progression (1-6). Ex vivo, this was successfully realized in flow cytometry (FC) by introducing cells into an artificial flow and by laser counting each cell after hydrodynamical focusing the cells in a flow chamber into a single-cell-file flow (8-11). To date, the FC is a well-established, powerful technique that has revolutionized cell diagnostics in vitro (10). Nevertheless, the extraction of cells from a living system and their preparation are time-consuming procedures leading to many artifacts, including the changes of cell-surface protein expression (12-15). In addition, it is extremely difficult to improve a cancer patient's survival when incurable metastases have already developed by the time of initial diagnosis with existing CTC assays. The sensitivity of these assasy ex vivo is limited by small sample volumes (2).

The use of vessels in living organisms as natural tubes with flowing cells in the native biological environments could improve detection sensitivity by assessment of the significantly larger blood or lymph volumes circulating in the peripheral vasculatures. The intravital microscopy demonstrated a potential to detect rare fluorescence-labeled cancer cells in the blood circulation (2,16,17); however, the main goal of flow cytometry to quantitate every single cell is not yet realized in vivo. In addition, the use of fluorescence labeling in vivo raises potential problems for translating this technology to use in humans: (1) the cytotoxicity of available fluoresecent tags; (2) the immune response to tags leading to phagocytic clearance of labeled CTCs; and (3) light scattering and the auto fluorescent background allowing the assessment of only superficial microvessels (depth, ≤0.3-0.5 mm) with a slow flow rate (2,16-19).

Recently we developed alternative approaches to in vivo FC based on the principles of photothermal (PT) and photoacoustic (PA) spectroscopy with near-infrared (NIR) pulse lasers, either without labeling or with conventional contrast agents (e.g., indocyanine green [ICG] or strongly absorbing gold nanoparticles (e.g., gold nanorods [GNRs]) and carbon nanotubes (CNTs) as advanced non-toxic PT and PA labels (16,20-26). This technique, which is inherent to light scattering, demonstrated an unprecedented sensitivity threshold of one cancer cell or bacteria in a background of 10⁸-10 ⁹ normal cells in the blood flow blood in vivo (23,24). However, single-cell detection in lymphatics still remains a challenge despite its clinical significance for early diagnosis of metastases, inflammation, immune disorders, lymphedema, and malformation, among many other diseases (27-33).

Here we introduce a new tool based on the principle of FC, an in vivo lymph test that noninvasively counts each cell in the lymph flow using two discoveries: 1) natural cell-focusing phenomenon in lymph vessels and 2) PA effects in single cells detectable with an ultrasound technique.

MATERIALS AND METHODS

Integrated lymph flow cytometer

The cytometer was built on the technical platform of an upright Olympus BX51 microscope (Olympus America, Inc.) with incorporated PA, PT, fluorescent, and transmission digital microscope (TDM) modules (Fig. 1). A tunable pulse laser parametric oscillator (OPO, Lotis Ltd., Minsk, Belarus) was used to irradiate cells at the following parameters: wavelength, 420-2,300 nm; pulse width, 8 ns; beam diameter, 10-50 μm, which can be adjusted in a linear configuration 6 μ wide and 50 μm long; fluence range, 1-10⁴ mJ/cm²; and pulse repetition rate, 10-50 Hz.

Figure 1

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

Laser-induced PA waves (referred to as PA signals) were detected with an ultrasound transducer (XMS-310, 10 MHz, Panametrics). After amplification (amplifier model 5662, Panametrics), signals were recorded with a Boxcar technique (Stanford Research Systems, Inc.) and a Tektronix TDS 3032B oscilloscope, and then analyzed with standard and customized software. Boxcar technique provided averaging of PA signals from cells and their time-resolved discrimination from background signals. The study was performed mostly in the visible-NIR spectral range of 450—950 nm, at which most cellular chromophores have distinctive, relatively narrow (20—40 nm) spectral fingerprints without significant interference from DNA, RNA, lipids, or water in the background, which absorb mainly in the ultraviolet (<350 nm) and the infrared (>950 nm) ranges.

In PT imaging (PTI) mode, a laser-induced temperature-dependent variations of the refractive index in cells the technique could were visualized with the use of a second, collinear probe laser pulse from a Raman shifter (wavelength, 639 nm; pulse width, 13 ns; pulse energy, 2 nJ, and delay time between pulses, 0—10 μs) and a CCD camera (AE-260E, Apogee Inc).

In PT thermolens mode, laser-induced refractive heterogeneity averaged over a whole cell caused the defocusing of a collinear He-Ne laser probe beam (model 117A, Spectra-Physics, Inc.; wavelength, 633 nm; 1.4 mW) and a subsequent reduction in the beam's intensity at its center detected with a photodiode (C5658; Hamamatsu Corp.).

High-resolution (300 nm) TDM, fitted with high-speed (10-40,000 frames per second [fps]) CMOS (MV-D1024-160-CL8; Photonfocus AG, Lachen, Switzerland) and high sensitive CCD (Cascade: 512; Photometrics, Roper Scientific, Inc.) cameras, was used to navigate laser beams, image fast moving cells, and verify the exposure of individual cells to the laser beam when the cells moved in single file.

A fluorescence module featuring a highly sensitive PentaMAX CCD camera with an intensifier (Princeton Instruments, Inc.) was used to verify the PA-PT data and identify labeled cells.

Animal models

The rats (Sprague-Dawley, weighing 150—200 g) and nude mice (nu/nu, weighing 20—25 g), both purchased from Harlan Sprague-Dawley were used in accordance with protocols approved by the UAMS Institutional Animal Care and Use Committee.

The experiments involved rat mesentery, which represents a near-ideal animal model for FC demonstration of proof of concept of a FC in vivo because it consists of very thin (8—17 μm), relatively transparent low-light-scattering tissue with a single layer of separate and clearly distinguished blood and lymph microvessels (Fig. 2a). After standard anesthesia with ketamine/xylazine, 50/10 mg/kg, i.m., the animals were laparotomized by a midabdominal incision, and the small-intestinal mesentery was placed on a customized, heated (37.7°C) microscope stage and suffused with warmed Ringer's solution (37°C, pH 7.4) containing 1% albumin to prevent protein loss. The ultrasound transducer was gently attached to the mesenteric tissue to prevent vessel compression.

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; (more ...)

The non-invasive animal model involved a mouse ear, which has a thin (~270 μm) and relatively transparent structure with a well-defined vasculature (Fig. 2b and c). The examined ear lymph vessels had diameters of 100—180 μ, flow velocity of 0.5—3 mm/s, and depth location of 50—150 μm. To detect cells, the anesthetized animals were placed on a warmed stage, and warm water was topically applied for acoustic matching of the transducer and the ear tissue.

Cells

The B16F10 mouse melanoma cells (ATCC) were cultured at 37^°C in a humidified atmosphere containing 5% CO₂ with Dulbecco's modified Eagle medium (Gibco-Invitrogen) supplemented by 10% fetal bovine serum.

Normal fresh cells were obtained from a heparinized whole blood sample of donor animals after terminal blood collection. Red blood cells (RBCs) were isolated by simple centrifugation (400×g for 10 min at room temperature), and white blood cells (WBCs), in particular, lymphocytes —were isolated by Histopaque gradient (Sigma) density gradient centrifugation, respectively as recommended by the supplier.

Photoacoustic-photothermal assessment of cells in vitro

WBCs, RBCs, and melanoma cells first underwent cell viability testing with the trypan blue exclusion test and were found to be 92-96% viable. Placed in phosphate-buffered saline (PBS), viable cells were then analyzed in 8.6-μl wells (S-24737, Molecular Probes) with a suspension thickness of 120μm.by time-resolved monitoring of linear and nonlinear PT-PA signals from single cells at different wavelengths. These data were verified by TDM and fluorescence imaging.

In vivo lymphatic mapping

The Evans blue (EB) stain, conventionally used for lymphatic research, was used in our investigation also as PA contrast agent to determine the location of lymphatic vessels. Specifically, 5 μl of 1% EB dye in 0.9% NaCl was injected intradermally into the ear tip with a 10-μl Hamilton solution syringe. PA mapping started 5 min after injection. Fluorescent images were obtained after the labeling of blood vessels in mouse ear with FITC-dextran (molecular weight [MW], 40 kDa; excitation/emission wavelengths, 492 nm/518 nm; green), and the labeling of lymph microvessels with tetramethylrhodamine isothiocyanate (RITC)-dextran (MW, 150 kDa; excitation/emission wavelengths, 495 nm/595 nm; red). In separate tests, fluorescein isothiocyanate (FITC)-dextran was injected into mesenteric lymphatic vessels with the FemtoJet microinjection system (Eppendorf North America).

For NIR mapping of lymphatic vessels, ICG, 0.2 ml/100 g of body weight, was injected into rat tail vein. A diode laser provided excitation at a wavelength of 805 nm and a fluence of 0.25 mW/cm²; re-emitted fluorescence was filtered at 830 nm and then detected with the CCD camera. The dye was observed to accumulate in mesenteric lymph microvessels 40-50 min after injection.

Melanoma model

Melanoma tumor in the ear of the mice was created by inoculated of 10⁶ B16F10 mouse melanoma cells in 50 μl of phosphate-buffered saline with a 30-gauge needle. The tumor growth was monitored on a daily basis by measuring tumor size (2 dimensions with calipers). On days 3, 5, 10, 20, and 30, CTCs were counted after inoculation for 1-3 hours in the lymph vessels near the primary tumor. To properly navigate the laser beam on the colorless ear lymphatics, the lymph vessels were visualized by local injection of EB dye (see above). The PA detection of melanoma cells was performed at laser wavelength 850 nm, where melanoma still had significant absorption (compared to maximum absorption in the visible range) without notable influence of the EB dye. That dye absorbs only in the visible spectral range up to 680 nm. On day 30 after inoculation (late stage of tumor development), CTCs were assessed at a distant site in a mesenteric lymph vessel. In parallel with PA detection , the mesenteric lymphatics were assessed by high-speed TDM imaging. Finally, the animals were euthanized, and tissue sections (cervical and mesenteric lymph nodes, liver, lung, kidney, brain, etc.) were examined by immunohistochemical staining for distant metastases. All specimens were prepared using the standard protocol provided by the manufacturer.

Imaging of B lymphocytes in vivo

To identify B lymphocytes, intravital immunophenotyping was used in analogy to the identification of cells with probes targeting specific antigens or molecular markers on the surface of a cell. In particular, the CD45 antibodies (Abs) specific to B lymphocytes were labeled with the fluorescent tag phycoerythrin (excitation/emission wavelengths, 488 nm/ 578 nm; BD PharMingen) and injected into rat peritoneum.

In vivo induction of apoptosis and immunophenotyping

Apoptosis of lymphocytes was intravitally induced by intraperitoneal injection of 1 μM dexamethasone (Sigma-Aldrich, St Louis, MO). After 6 hrs, the apoptotic cells were labeled in vivo by intraperitoneal injection of Annexin-FITC complex (emission - 480 nm; exitation - 530 nm).

Two-wavelength two- pulses mode for irradiation of multicolor probes

Two-wavelength irradiation of cells in vivo was performed with the setup described above (Fig. 1), with an increase to 10 μJ in the energy of the probe pulse from the Raman shifter (639 nm). The two laser pulses had the same repetition rate (50 Hz) and following different parameters, first pulse: a tunable wavelength in the range of 420-2,300 nm, a pulse width of 8 ns; and second pulse: a fixed wavelength of 639 nm, a pulse width of 12 ns, and a tunable delay (0-10 μs) to the first pulse. Two-wavelength mode was used for real-time selective detection of (1) necrotic lymphocytes labeled with GNRs, (2) apoptotic lymphocytes labeled with gold nanoshells (GNSs), and (3) neutrophils labeled with CNTs. The GNRs had a diameter of 15 nm and a length of 40 nm with a relatively narrow absorption band of 75 nm near 640 nm; they were synthesized according to an established protocol (25). GNSs with 120-nm in diameter with a maximum absorption near 860 nm were provided by Nanospectra Bioscience, Inc. Single-walled CNTs measuring on average 168 nm long and 1.7 nm in diameter had monotonically decreased absorption with increases in the wavelength (similar to that for melanoma cells); they were purchased from Carbon Nanotechnologies Inc. Apoptosis of lymphocytes was induced in vitro by 2-h incubation with 1 μM staurosporine (Sigma-Aldrich) at 37°C and controlled with the Annexin-VFLUOS staining kit (Roche Applied Science). Necrosis of lymphocytes was induced by ethanol (70%, 5 min). Aliquots (100 μl) of cells in phosphate-buffered saline were incubated with 100 μl of CNTs, GNRs, and GNSs for 15 min at 37°C room temperature.

Statistical analysis

Results are expressed as means plus/minus the standard error of at least three independent experiments (P <0.05). Statistica 5.11 software (StatSoft, Inc.) and MATLAB 7.0.1 (MathWorks) were used for the statistical calculations.

RESULTS

Natural cell-focusing phenomenon

Adaptation of the FC principles to in vivo studies requires precautions related to light scattering by surrounding tissues, and to fluctuations in the position and velocity of cells in a vessel, especially in the “colorless” lymphatics with unstable, turbulent, slower, and oscillating flow (17, 34). The schematic (Fig. 3a) is derived from our long-term observation of lymphatic function in many (110) rats (17). Lymph motion results from phasic contractions of the lymphangion, the segment of lymph vessels situated between two valves that undergoes cycles of compression (systole) and dilation (diastole) (17,29,30). During systole, phasic contractions create a positive pressure gradient (ΔP = P₁ - P₂ ≥ 0) (Fig. 3b) that moves lymph forward for 0.5-2 sec; during diastole, the pressure gradient decreases to 0, and lymph flow stops for 0.05-0.1 sec; and with a negative pressure gradient, lymph motion is retrograde for 0.2 ± 0.02 sec (i.e., approximately 12 times shorter in duration than forward motion). Lymph flow then stops again for a longer time, 1-3 sec, and the lymphangion fills with lymph from its distal portion. In this stage, the spatial distribution of cells (mainly WBCs) is random, and cells fluctuate in the radial directions up to 70-100 μm. At the end of diastole, pressure gradient ΔP become positive, the valve begins to open, and lymph moves forward again (Fig. 3b). This complex cell motion makes it difficult to detect individual cells compared to FC in vitro with the well-organized single-file flow by hydrodynamic cell focusing using sheath fluids created by two coaxial tubes acting as artificial nozzle (9-11). Nevertheless, we discovered that nature has also created single-file cell flow in a localized zone near the valve. In particular, in vessels with diameters of 136 ± 10 μm, the valve leaflets formed natural nozzles, measuring approximately 44±7 μm in diameter, that provided an approximately threefold constriction of flow. This constriction significantly increased flow velocity, on average from 300-500 μm/s up to a few mm per second. As a result, cell acceleration, together with sheering forces, led to hydrodynamic focusing of cells into single-file cell flow, with radial cell fluctuations of just 5-10 μm (Fig. 3c). The phasic contraction in the central part of the lymphangion between valves provides also secondary cell focusing (Fig. 3d); however, due to the relatively low degree of constriction (20-30%), single-file cell flow was only observed in small-diameter vessels ( <80 μm). We also discovered laser-induced localized squeezing of a lymph vessel (Fig. 3e). At laser parameters indicated in figure caption this effect was reversible, with a vessel-recovery time of 5-10 min.

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× (more ...)

Schematic of integrated lymph flow cytometer

The discovery of natural, hydrodynamic focusing of cells led us to develop an advanced lymph PA flow cytometer (PAFC) (Fig. 3a), in which individual cells are irradiated near a valve nozzle when they form single-file flow. To verify this concept, we integrated PAFC with well-established TDM, fluorescence, and PT techniques, which also made PAFC more universal for analyzing cells with different absorption, fluorescence, or scattering properties. Navigation of the laser beam on colorless lymphatic vessels (Fig. 2a) was based on choosing a contrast agent whose spectral range of absorption, for example, in the visible-light range, did not interfere with cell absorption in the NIR range (e.g., with absorption from unlabeled melanoma cells or cells labeled with NIR nanoparticles). As results, PAFC provided detection of cells and simultaneous visualization of lymph with a standard tags, such as FITC (Fig. 2b and d), EB dye (Fig. 2c) and ICG (Fig. 2e). In particular, fast scanning (second scale) of a focused laser beam across the ear tissue that was stained with EB within the field of interest provided PA mapping/visualization of lymph vessels at 639 nm, where absorption of EB was still significant. In addition, interference with blood at 639 nm was minimal because blood absorption dropped to a level that was almost one-order magnitude less compared to the maximum absorption near 580 nm. This may make it possible to determine the lymph vessels' margins at a low concentration of EB (or when a vessel has a deep location), which is invisible with the naked eye.

The strong focusing of laser beam into the vessels on area near a valve nozzle provided selective detection of signals from cells preferentially in this area when necessary because a defocused relatively large laser beam generates smaller a PA signals than focused small diameter beam from the same targets.

Counting of metastastatic melanoma cells

PA detection of melanoma cells was based on laser-induced PT and accompanied PA effects in melanin as an intrinsic cell marker. To optimize the detection parameters, we applied in vitro PA and a PT technique, which provided signal-to-noise ratios in the range of 10-50 depending on cell “absorption” heterogeneity, and specific PT image signatures associated with cellular chromophore (pigment) distribution, such as cytochromes in lymphocytes, hemoglobin in RBCs, and melanin in melanoma cells (Fig. 4, third column). PT signals from single cells in a linear mode without cell photodamage (24) demonstrated the standard fast-rising unipolar peak associated with rapid (ns scale) cell heating and a slower (μs scale) tail corresponding to cell cooling ((Fig. 4, right columns). The PA signal from the same cells had a bipolar shape that was transformed into a pulse train due to reflection and resonance effects (Fig. 5a, b, top). The signals were compressed either at a slow (ms scale) oscilloscope rate or after signal acquisition and presented as vertical unipolar positive lines (Fig.5 a,b, bottom).

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 (more ...)

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 (more ...)

Because melanin has a broad absorption spectrum with a slight absorption decrease in the NIR spectral range, melanoma cells were detected at a wavelength of 850 nm, with simultaneous laser navigation on a lymph vessel using EB dye, which absorbs in the visible-light range up to 660 nm (Fig. 2c). Specifically, right position of laser beam on lymph vessel was adjusted when the PA signal amplitude reached its maximum at the laser wavelength of 639 nm.

In mice, early melanoma metastatic cells were observed to appear in a lymphatic vessel of the mouse's ear on the first week after inoculation, with further gradual increase a rate over the course of the next three weeks (1.1 ± 0.4 cells/min at second week ) (Fig. 5a). Routine examination of the sentinel lymph node (the first location of metastasis) by immunohistochemistry demonstrated no metastasis at the second week, one micrometastasis at the third week, and a few micrometastasis on the fourth week in these nodes as well as in the liver and lung (Fig. 2g). In mice with a late melanoma tumor in the ear (fourth week after tumor inoculation), metastatic cells appeared in distant (mesenteric) lymphatics at a rate 8.2 ± 1.07 cells/min. The presence of live and necrotic (partly destroyed) melanoma cells was confirmed by transmission imaging (Fig. 6g, and Fig. 6h, respectively). In selected experiments, the PA technique was able to detect single micrometastases in sentinel lymph nodes in vivo on the second week after inoculation.

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×, (more ...)

Label-free PA detection of lymphocytes

Spectroscopic studies in vitro revealed that PA signals from lymphocytes reached maximal amplitude in the visible-spectral range near 530-550 nm associated with absorption by cytochromes mainly cytochrome c as the intrinsic absorption marker. Background PA signals from vessels and surrounding tissues were approximately 4-6-fold less than from single lymphocytes at this wavelength due to the low level of background absorption and laser focusing effects. The obtained cell rate was 60 ± 12 cells/min; cell heterogeneity resulted in 2-2.5-fold fluctuations in PA signal amplitude from cell to cell. We also observed rare signals (~1%) that had strong amplitudes exceeding those of lymphocyte signals by 6-15 times (Fig. 5b); spectral and imaging (Fig. 4) analysis identified rare single RBCs as the sources of these signals.

Real-time two-wavelength lymph flow cytometry with multicolor probes

Because of the relatively short (10^-2-10^-3 sec) time during which cells in the lymph flow appear in the detected volume after the valve nozzle, existing time-consuming imaging and spectral scanning methods are not quite adequate for multispectral in vivo PA flow cytometry with pulse lasers. We introduced a novel approach for real-time multispectral exposure of fast-moving cells labeled with mucticolor probes with experimental proof for two-wavelength modes and three nanoparticle types.

Necrotic and apoptotic lymphocytes, and live neutrophils and were labeled with GNSs, GNRs, and CNTs, respectively. The GNSs and, especially GNRs and had relatively narrow absorption bands, 180 nm and 75 nm (at a 50% amplitude level) with maximum absorption at 860 nm and 640 nm, respectively; in contrast, the absorption spectrum of CNTs was relatively broad covering visible and NIR range (35). These labeled cells, mixed in equal proportions, were injected into a mouse tail vein. After 6 hrs, we observed in mesenteric lymphatics irradiated with two laser pulses at wavelengths of 865 nm and 639 nm with a 10-μs delay between the pulses rare a PA signals (1-3/min) associated with cells labeled by different nanoparticles. In particular, PA signals from necrotic lymphocytes were generated by a laser pulse at 639 nm only after a 10-μs delay (Fig. 5c), while PA from apoptotic lymphocytes were generated by laser pulse at a wavelength of 865 nm with no delay between the pulses (Fig. 5d). Live neutrophils yielded two PA signals with a 10-μs delay because of comparable CNT absorption at both 639- and 865-nm wavelengths (Fig. 5e).

High-resolution single cell imaging

The high-speed TDM provided in vivo label-free visualization of natural single cells transport from tissue to regional (i.e., sentinel) lymph nodes (Fig. 6), including monitoring of their passage through the endothelial wall to an initial lymphatic vessel (Fig. 6b-c). On the basis of notable differences in size and shape, TDM provided the identification of individual leukocytes (Fig. 6d), RBCs (Fig. 6e), and (Fig. 6f) and melanoma cells (Fig. 6g,h) in lymph flow. In particular, in relatively transparent mesenteric lymphatics, the metastatic cells were identified by their larger size (approximately two-times that of leukocutes) and black localized pigmentation (Fig. 6g). Melanoma cells had round shapes, which were likely in a live stage, or an irregular form that was probably in the necrotic stage (Fig. 6h). They were transported with a velocity similar to normal cells (around 1 mm/sec). In contrast to lymphocytes, which were moved typically as single cells, melanoma cells traveled frequently as small aggregates.

To count lymphocyte`s subsets (e.g., B lymphocytes), the fluorescence-labeled antibodies specific to CD45 markers of B lymphocytes were injected into mouse peritoneum in vivo followed by time-resolved fluorescence monitoring of mesenteric lymphatics (Fig. 6i). The mean velocity of B lymphocytes in lymph flow was determined to be 720 ± 18 μm/s, and the proportion of B lymphocytes in prenodal lymph was 4.5%.

In order to capture an image of apoptotic cells, apoptosis was induced by intraperitoneal injection of dexamethasone followed 6 hrs later by the injection of Annexin V-FITC and then by fluorescence monitoring of apoptotic cells. The majority of apoptotic lymphocytes were found mainly in mesenteric tissues. We observed also a single lymphocytes adhered to a vessel twall. Only a few apoptotic cells were detected in lymph flow during the 1-h observation (Fig. 6j).

DISCUSSION

Compared to the blood test, the lymphatics has been paid much less attention, although recent discoveries in the biology of lymphatics (e.g., new receptors, LYVE-1, VEGF-3, and PROX1) emphasized its the key role in the development of many specific diseases such as metastasis, infections, or immunity disorders (27,28). To date, many questions of lymphatic function are still not quite answered. For example, despite dramatically growing evidence of the crucial role of the lymphatic route in the spread of metastatic cells such as melanoma or breast cancer to other organs, researchers knew almost nothing about transport of these cells through lymph vessels before their invasion into lymph nodes (27,28). Another very important issue is lymph transportation of immune-related cells. According to a few in vitro animal studies, lymphatics are mainly composed of immune system-related cells: 85% T lymphocytes, 5% B lymphocytes, and 10% other cell types (dendritic, and macrophages, among others) (31,32). Besides differences in composition, the total concentration of cells in lymphatics is lower and varies significantly (10³-10⁵ cells/μl) than in the blood flow (10⁸-10⁹cells/μl). Since, the sampling of lymph is not well-established low-accurate procedure, the exact composition of cells in lymphatics (e.g., amount RBCs), as well as cell rate, especially, at pathology, is not still clear. Despite significant progresses in diagnostic techniques (e.g., magnetic resonance imaging, lymphoscintography, or fluorescence lymphography), to date, none has demonstrated the ability to real-time detection of individual cells in the lymph flow.

As we present here, PAFC using a cell focusing phenomenon and PA effect into single cells has potential to address to this and many other related issue, in particular, lymph testing in vivo. Discovered in 1860 PA effects, as generation of sound by absorption of light, recently has been used for biomedical imaging of biotissue consisted of many cells only (36). We demonstrated here, that endogenous cellular absorption can serve as intrinsic cell-specific markers having distinguished spectral and spatial signatures and its level is enough to produce sound in single cells readable with conventional ultrasound technique. This is non-invasive procedure because required a laser fluence is in the range of below 34-100 mJ/cm², which is the ANSI laser safety standard at the spectral range of 800-1100 nm, respectively (37). The absorption-sensitivity threshold of the PAFC at the single-cell level was estimated to be 10^-2 cm^-1, that is, at least four orders of magnitude better than that of absorption spectroscopy for the same condition. This high sensitivity can be further enhanced during interaction of pulse laser with absorbing nanoclusters associated with self-assembly of endogenous chromophores (e.g., cytochromes and melanin) or exogenous nanoparticles. Laser-induced optical, thermal, acoustic, and accompanied phenomena from individual closely located chromophore molecules or nanoparticles into clusters may overlap and provide synergistic amplification of PT/PA signals (38). Because many cellular structures are weakly fluorescent in native state and may create molecular assemblies/clusters, laser energy absorbed by these clustered structures is transformed mostly into heat and then into amplified sound, that makes PA “listening” of individual cells is promising for cell biology as alternative (or supplementary) to fluorescent “seeing” them. As a result, the high absorption-sensitivity of PAFC together with low background absorption in colorless lymphatics (compared to blood vessels) provides the opportunity for label-free identification and quantitation of RBCs, melanoma cells, and different types of WBCs, (Fig. 4) including immunity-related cells in normal, apoptotic, and necrotic stages. In particular, PAFC revealed the presence of low concentrations of RBCs in lymphatics, which may have diagnostic value for evaluation some diseases , in particular, venous disfunctions, or diabetes accompinined by increasing blood vessel permeability (33,39). The label-free identification of a subpopulation of WBCs as well as other cancer cells (e.g., breast, or prostate) is not realized yet. However, it should be possible, on the basis of spectral fingerprints of the different cytochromes, which concentration and mutual proportion may vary during cell metabolism, apoptosis, or carcinogenesis (40).

The most significant achievement is a real-time label-free detection of metastatic melanoma cells in lymph flow. PAFC at a WBC rate of 10² cell/s in vessels with a diameter of 200 μm is able to detect just one melanoma cell during 3 h of observation; this rate is approximately equivalent to one melanoma cell in the background of 10⁶ WBCs. This unprecedented sensitivity threshold in lymphatics in vivo is unachievable with any other existing technique. In larger lymph vessels, the sensitivity threshold and the observation time should be improved because of the higher WBC rate. To proof of concept, we used superficial lymphatic vessels at a depth of 100-200 μm; however, recent advances with PA imaging of blood vessels 5-6 cm deep (36) support the potential of the new tool to sense lymph vessels and lymph nodes at depths of a few centimeters with an expected resolution of 0.1-0.3 mm with NIR laser and focused cylindrical ultrasound transducers (25,26). We presented a t few data of detection metastasis in sentinal lymph nodes (SLNs ), because this topic is the outside scope of this paper. Nevertheless, based on these data, we can suggest a potential of PAFC for early detection metastasis in vivo with a sensitivity that is superior to existing conventional assays ex vivo. In general, for the first time, we obtained in vivo evidence that (1) lymph vessels transport live metastatic cells and remove destructed melanoma cells; (2) melanoma cells travel in lymph flow preferentially as small aggregates; (3) the lymphatic route involves the earliest stage of metastatic disease, even before detectable metastasis in SNLs are developed; and (4) the lymphatic system can promote dissemination of tumor cells at the late stage of metastatic disease by passing cells to distant organs (e.g. melanoma cells from an ear tumor may be spread to mesenteric lymphatics within the abdominal cavity). We believe this PA visualization of “colorless” lymphatics and, especially, SNLs using conventional blue dyes (e.g., Evans blue, Methylen blue, or Lymphazurin) that absorb in the visible spectral range without interference with cell detection in the NIR range may help resolve complex and relatively unexplored issues, including integration in vivo non-invasive SLN mapping, single-cell detection, and metastasis assessment. This includes also the diagnostic value of lymphogenously disseminated rare CTCs for metastasis development (1,27).

The specificity of PAFC can be enhanced by the use of multicolor functionalized nanoparticles with different absorption spectra as we demonstrated for selective detection of neutrophils, and necrotic and apoptotic lymphocytes in lymphatics. These nanoparticles, and especially gold-based probes have shown recently excellent potential for optical labeling because they do not photobleach or blink, are photostable, strong absorbers, can easily be conjugated with many proteins and Abs (38). Furthermore, compared to conventional organic labels, gold is nontoxic, has excellent biocompatibility, and has been used in humans for 50 years (e.g., dental prosthetics, implants, and treatment of arthritis). The gold-based nanoparticle was recently approved for clinical trials, including PT therapy of cancer (41). We believe that the in vivo lymph test concept may be further developed to extend its utility beyond diagnosis to include the possibility of in vivo lymph purging by elimination, at the appropriate laser energy level, of cancer cells labeled with nanoparticles in flow without harmful effects on surrounding normal cells and tissue.

To verify the concept of the in vivo rapid multispectral lymph test, we applied a two-wavelength mode, although the potential exists to increase the number of spectral channels to include low-cost, compact diode lasers with different wavelengths and high-pulse repetition rates (up to 10-50 kHz). Assuming the appearance of cells in the irradiated volume approximately during 1 ms (for a flow velocity of 5 mm/s and laser beam diameter of 5 μm) and a minimum delay of 10 μs between laser pulses, approximately 10^-3 sec/10^-5 sec = 100 laser pulses with 100 different wavelengths can be used for spectral cell identification ( in modern FC only 8-16 wavelength).

The integrated PA and fluorescent lymph flow cytometry schematics provided real-time detection of individual lymphocytes in norm and apoptosis by their labeling with conventional fluorescent probes in vivo (Fig. 5i,j). We did not indicate notable changes in lymphatics function parameters (17) at used doses that were in line with other data (2,16), although a possible toxicity should be further estimated before use in clinics. We are confident that replacement of fluorescent dyes on PA gold nanoparticles could solve this problem. In addition, conventional fluorescent labels can be used as PA/PT labels because the limited quantum yield of most of them, typically in the range of 1-20%, results in the transformation of most absorbed energy into heat (25). The PA/PT technique is very sensitive to this heat, and this high PA sensitivity makes it possible to use labeling agents at lower concentrations than standard doses, which may reduce the risk of cytotoxicity.

The phasic contractions of lymph vessels acting as a natural concentrator of flowing cells near a vessel's center in combination with lymphatic valves acting as natural nozzles provided cell positioning with minimum radial fluctuation at level 5-10 μm in the lymph vessel with diameter of 150-200 μm. It was sufficient for counting each cells near valve exit using linear laser beam shape with length 40-70 μm and width 5-15 μm.

To exclude counting the same cells during their backward motion, we applied the monitoring cell motion with TDM with further exclusion of data obtained for backward cell motion. Besides, the contractions in some lymphangions and cycles of valve activity (opening-closing) were found to be relatively stable, with an average rate of 12 ± 1/min and 9 ± 1/min, correspondingly (17). So laser exposure was adjusted to these particular rhythms. In addition, detection of forward moving cells only can be achieved by synchronization of laser exposure with the vessel-wall or valve motion coinciding to forward cell motion, controlled with a pilot laser.

The majority of examined lymphangions had well-developed bicuspid funnel-shaped valves and operated mainly under viscous fluid forces. This anatomic structure was not quite optimal for creation of the natural small nozzles with clear margins. In addition, some external factors that are not well controlled, such as muscle contractions, respiratory movements, and intestinal peristalsis, can influence valve function and phasic constrictions. Nevertheless, most selected active valves provided excellent natural hydrodynamic focusing of cells into single-file flow in analogy with artificial hydrodynamic focusing in conventional FC in vitro. We believe that a similar approach using vein valves for cell focusing can also be applied to FC of blood flow.

Reversible laser-induced short term lymph vessel squeezing (Fig. 2e) is also very attractive for the purpose of forming single-file cell flow, although its mechanism (e.g., laser activation of pacemakers, or smooth muscle), possible invasive nature, side effects, and control degree require further study. In conclusion, a novel combination of PA microscopy/spectroscopy and FC with many innovations (natural cell focusing in vivo, PA effects in single cells, endogenous absorption as an intrinsic cell signature, real-time multispectral identification of cells with multicolor nanoparticles, combined navigation/detection schematics using for visualization of “colorless” lymphatics the contrast agents without interference with cell detection; an almost ideal preclinical animal model to test the concept of lymph FC in vivo; and integration of PA, PT, fluorescent, and TDM techniques, which makes the in vivo lymph test more universal) may be considered a new, powerful tool in lymphatic research with a broad spectrum of potential applications including real-time detection of various types of cells (e.g., RBCs, lymphocytes, dendritic, metastatic, infected, inflamed, and stem cells) at different states (e.g., norm, apoptotis, or necrosis), infectious agents such as bacteria and viruses, contrast agents, nanoparticles, liposomes, and drugs in a variety of lymphatic vessels (e.g., initial, afferent, and efferent lymphatics, and lymph nodes) under normal and pathological conditions. (e.g., tumors, infections, lymphedema). A quick translation of this technology to use in humans is anticipated with the development of a portable device that can be attached to lymph vessels and nodes to single cell diagnostics, metastasis detection, or assessment of therapy (i.e., radio- and chemosensitivity testing) through the quantification of metastatic and apoptotic cells, or different impacts (e.g., nicotine) (42).

ACKNOWLEDGMENTS

The authors thank V. Kalchenko, PhD of Weizmann Institute of Science, Rehovot, Israel, for providing Fig. 2f. We would like to thank Scott Ferguson for assistance with the laser measurements, and Nanospectra Biosciences, Inc, for providing gold nanopartilces. We also thank the Office of Grants and Scientific Publications at UAMS for editorial assistance during the preparation of this manuscript.

This work was supported by the National Institutes of Health (NIH) and by the NIH/National Institute of Biomedical Imaging and Bioengineering under grants EB000873 and EB0005123, and by the Arkansas Biosciences Institute.

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