Effect of pulsed laser parameters on photoacoustic flow cytometry efficiency in vitro and in vivo.

Photoacoustic flow cytometry is one of the most effective approaches to detect "alien" objects in the bloodstream, including circulating tumor cells, blood clots, parasites, and emboli. However, the possibility of detecting high-amplitude signals from these objects against the background of blood depends on the parameters of the laser pulse. So, the dependencies of photoacoustic signals amplitude and number on laser pulse energy (5-150 µJ), pulse length (1, 2, 5 ns), and pulse repetition rate (2, 5, 10 kHz) for the melanoma cells were investigated. First, the PA responses of a melanoma cell suspension in vitro were measured to directly assess the efficiency of converting laser light into an acoustic signal. After it, the same dependence with the developed murine model based on constant rate melanoma cell injection into the animal blood flow was tested. Both in vivo and in vitro experiments show that signal generation efficiency increases with laser pulse energy above 15 µJ. Shorter pulses, especially 1 ns, provide more efficient signal generation as well as higher pulse rates. A higher pulse rate also provides more efficient signal generation, but also leads to overheating of the skin. The results show the limits where the photoacoustic flow cytometry system can be effectively used for the detection of circulating tumor cells in undiluted blood both for in vitro experiments and for in vivo murine models.

Complex genetic, photothermal, and photoacoustic analysis of nanoparticle-plant interactions.

Understanding the nature of interactions between engineered nanomaterials and plants is crucial in comprehending the impact of nanotechnology on the environment and agriculture with a focus on toxicity concerns, plant disease treatment, and genetic engineering. To date, little progress has been made in studying nanoparticle-plant interactions at single nanoparticle and genetic levels. Here, we introduce an advanced platform integrating genetic, Raman, photothermal, and photoacoustic methods. Using this approach, we discovered that multiwall carbon nanotubes induce previously unknown changes in gene expression in tomato leaves and roots, particularly, up-regulation of the stress-related genes, including those induced by pathogens and the water-channel LeAqp2 gene. A nano-bubble amplified photothermal/photoacoustic imaging, spectroscopy, and burning technique demonstrated the detection of multiwall carbon nanotubes in roots, leaves, and fruits down to the single nanoparticle and cell level. Thus, our integrated platform allows the study of nanoparticles' impact on plants with higher sensitivity and specificity, compared to existing assays.

Photothermal multispectral image cytometry for quantitative histology of nanoparticles and micrometastasis in intact, stained and selectively burned tissues.

There is a rapidly growing interest in the advanced analysis of histological data and the development of appropriate detection technologies in particular for mapping of nanoparticle distributions in tissue in nanomedicine applications. We evaluated photothermal (PT) scanning cytometry for color-coded imaging, spectral identification, and quantitative detection of individual nanoparticles and abnormal cells in histological samples with and without staining. Using this tool, individual carbon nanotubes, gold nanorods, and melanoma cells with intrinsic melanin markers were identified in unstained (e.g. sentinel lymph nodes) and conventionally-stained tissues. In addition, we introduced a spectral burning technique for histology through selective laser bleaching areas with nondesired absorption background and nanobubble-based PT signal amplification. The obtained data demonstrated the promise of PT cytometry in the analysis of low-absorption samples and mapping of various individual nanoparticles' distribution that would be impossible with existing assays. Comparison of PT cytometry and photoacoustic (PA) cytometry previously developed by us, revealed that these methods supplement each other with a sensitivity advantage (up to 10-fold) of contactless PT technique in assessment of thin (≤100 µm) histological samples, while PA imaging provides characterization of thicker samples which, however, requires an acoustic contact with transducers. A potential of high-speed integrated PT-PA cytometry for express histology and immunohistochemistry of both intact and stained heterogeneous tissues with high sensitivity at the zepromolar concentration level is further highlighted.

Ultra-fast photoacoustic flow cytometry with a 0.5 MHz pulse repetition rate nanosecond laser.

In vivo photoacoustic (PA) flow cytometry (PAFC) has great potential for detecting disease-associated biomarkers in blood and lymph flow, as well as real-time control of the efficacy of photothermal (PT) and other therapies through the counting of circulating abnormal objects. We report on a high speed PAFC with a Yb-doped fiber laser having a 0.5-MHz pulse repetition rate at a wavelength of 1064 nm, pulse width of 10 ns, and energy up to 100 microJ. This is the first biomedical application of PA and PT techniques operating at the highest pulse repetition rate of nanosecond lasers that provide 100-fold enhancement in detection speed of carbon nanotube clusters, as well as real-time monitoring of the flow velocity of individual targets through the width of PA signals. The laser pulse rate limits for PT and PA techniques depending on the sizes of laser beam and targets and flow velocity are discussed. We propose time-overlapping mode and generation of periodic nano- and microbubbles as PA-signal and PT-therapy amplifiers, including discrimination of small absorbing targets among large ones. Taking into account the relatively low level of background signals from most biotissues at 1064 nm, our data suggest that a nanosecond Yb-doped fiber laser operating at high pulse repetition rate could be a promising optical source for time-resolved PA and PT cytometry, imaging, microscopy, and therapy, including detection of nanoparticles and cells flowing at velocities up to 2.5 m/s.

Photothermal and photoacoustic Raman cytometry in vitro and in vivo.

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump-Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420-2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level.

In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells.

The spread of cancer cells between organs, a process known as metastasis, is the cause of most cancer deaths. Detecting circulating tumour cells -- a common marker for the development of metastasis -- is difficult because ex vivo methods are not sensitive enough owing to limited blood sample volume and in vivo diagnosis is time-consuming as large volumes of blood must be analysed. Here, we show a way to magnetically capture circulating tumour cells in the bloodstream of mice followed by rapid photoacoustic detection. Magnetic nanoparticles, which were functionalized to target a receptor commonly found in breast cancer cells, bound and captured circulating tumour cells under a magnet. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. By integrating in vivo multiplex targeting, magnetic enrichment, signal amplification and multicolour recognition, our approach allows circulating tumour cells to be concentrated from a large volume of blood in the vessels of tumour-bearing mice, and this could have potential for the early diagnosis of cancer and the prevention of metastasis in humans.

In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser.

The circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. However, its clinical utility for metastasis prevention remains unclear, because metastases may already be present at the time of initial diagnosis with existing assays. Their sensitivity ex vivo is limited by a small blood sample volume, whereas in vivo examination of larger blood volumes may be clinically restricted by the toxicity of labels used for targeting of CTCs. We introduce a method for in vivo photoacoustic blood cancer testing with a high-pulse-repetition-rate diode laser that, when applied to melanoma, is free of this limitation. It uses the overexpression of melanin clusters as intrinsic, spectrally-specific cancer markers and signal amplifiers, thus providing higher photoacoustic contrast of melanoma cells compared with a blood background. In tumor-bearing mouse models and melanoma-spiked human blood samples, we showed a sensitivity level of 1 CTC/mL with the potential to improve this sensitivity 10(3)-fold in humans in vivo, which is impossible with existing assays. Additional advances of this platform include decreased background signals from blood through changes in its oxygenation, osmolarity, and hematocrit within physiologic norms, assessment of CTCs in deep vessels, in vivo CTC enrichment, and photoacoustic-guided photothermal ablation of CTCs in the bloodstream. These advances make feasible the early diagnosis of melanoma during the initial parallel progression of primary tumor and CTCs, and laser blood purging using noninvasive or hemodialysis-like schematics for the prevention of metastasis.

Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents.

Carbon nanotubes have shown promise as contrast agents for photoacoustic and photothermal imaging of tumours and infections because they offer high resolution and allow deep tissue imaging. However, in vivo applications have been limited by the relatively low absorption displayed by nanotubes at near-infrared wavelengths and concerns over toxicity. Here, we show that gold-plated carbon nanotubes-termed golden carbon nanotubes-can be used as photoacoustic and photothermal contrast agents with enhanced near-infrared contrast ( approximately 10(2)-fold) for targeting lymphatic vessels in mice using extremely low laser fluence levels of a few mJ cm(-2). Antibody-conjugated golden carbon nanotubes were used to map the lymphatic endothelial receptor, and preliminary in vitro viability tests show golden carbon nanotubes have minimal toxicity. This new nanomaterial could be an effective alternative to existing nanoparticles and fluorescent labels for non-invasive targeted imaging of molecular structures in vivo.

In vivo fiber-based multicolor photoacoustic detection and photothermal purging of metastasis in sentinel lymph nodes targeted by nanoparticles.

This report introduces a novel diagnostic and therapeutic platform for in vivo non-invasive detection and treatment of metastases in sentinel lymph nodes (SLNs) at single cell level using an integrated system of multicolor photoacoustic (PA) lymph flow cytometry, PA lymphography, absorption image cytometry, and photothermal (PT) therapy. A melanoma-bearing mouse model was used to demonstrate the capability of this platform for real-time lymphatic mapping, counting of disseminated tumor cells (DTCs) in prenodal lymphatics, and detecting metastasis in SLNs and its purging. The detection and ablation of non-pigmented breast cancer cells in SLNs was achieved by labeling them with nanoparticles. The association between DTC count and SLN metastasis progression supports lymphatic DTCs as a novel prognostic marker of metastasis. The fiber-based portable PA device may replace the conventional SLN(s) excision and histology-based staging. The earliest detection of DTCs in the lymphatic vessels before the establishment of nodal metastasis may prevent metastasis by well-timed ablation of DTCs.

Quantum dots as multimodal photoacoustic and photothermal contrast agents.

Quantum dots (QDs) have primarily been developed as fluorescent probes with unique optical properties. We herein demonstrate an extension of these QD utilities to photoacoustic (PA) and photothermal (PT) microscopy, using a nanosecond pulse laser excitation (420-900 nm, 8 ns, 10(-3)-10 J/cm(2)). The laser-induced PA, PT and accompanying bubble formation phenomena were studied with an advanced multifunctional microscope, which integrates fluorescence, PA, PT imaging, and PT thermolens modules. It was demonstrated that QDs, in addition to being excellent fluorescent probes, can be used as PA and PT contrast agents and sensitizers, thereby providing an opportunity for multimodal high resolution (300 nm) PA-PT-fluorescent imaging as well as PT therapy. Further improvements for this technology are suggested by increasing the conversion of laser energy in PT, PA, and bubble phenomena in hybrid multilayer QDs that have optimized absorption, thermal, and acoustic properties.

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

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.

Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo.

The goal of this work is to develop in vivo photoacoustic (PA) flow cytometry (PAFC) for time-resolved detection of circulating absorbing objects, either without labeling or with nanoparticles as PA labels. This study represents the first attempt, to our knowledge, to demonstrate the capability of PAFC with tunable near-infrared (NIR) pulse lasers for real-time monitoring of gold nanorods, Staphylococcus aureus and Escherichia coli labeled with carbon nanotubes (CNTs), and contrast dye Lymphazurin in the microvessels of mouse and rat ears and mesenteries. PAFC shows the unprecedented threshold sensitivity in vivo as one gold nanoparticle in the irradiated volume and as one bacterium in the background of 10(8) of normal blood cells. The CNTs are demonstrated to serve as excellent new NIR high-PA contrast agents. Fast Lymphazurin diffusion in live tissue is observed with rapid blue coloring of a whole animal body. The enhancement of the thermal and acoustic effects is obtained with clustered, multilayer, and exploded nanoparticles. This novel combination of PA microscopy/spectroscopy and flow cytometry may be considered as a new powerful tool in biological research with the potential of quick translation to humans, providing ultrasensitive diagnostics of pathogens (e.g., bacteria, viruses, fungi, protozoa, parasites, helminthes), metastatic, infected, inflamed, stem, and dendritic cells, and pharmacokinetics of drug, liposomes, and nanoparticles in deep vessels (with focused transducers) among other potential applications.

Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters.

BACKGROUND AND OBJECTIVES: Unique properties of carbon nanotubes (CNTs) would open new avenues for addressing challenges to realize rapid and sensitive antimicrobial diagnostics and therapy for human pathogens. In this study, new CNTs' capabilities for photothermal (PT) antimicrobial nanotherapy were explored in vitro using Escherichia coli as a model bacterium. STUDY DESIGN/MATERIALS AND METHODS: Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) were incubated with E. coli K12 strain. CNTs' locations in bacteria and laser-induced thermal and accompanied effects around CNTs were estimated with TEM and PT microscopy, respectively. Multi-pulse lasers at 532 and 1064 nm with 12-ns pulse duration were used for irradiating sample mixtures at different laser fluences. Cell viability was evaluated using a bacterial viability test kit and epi-fluorescence microscopy. RESULTS: This study revealed CNTs' high binding affinity to bacteria, their capability to self-assemble as clusters at bacteria surfaces, and their inherent near-infrared (NIR) laser responsiveness. Cell viability was affected neither by CNTs alone nor by NIR irradiations alone. Notable changes in bacteria viability, caused by local thermal and accompanied bubble-formation phenomena, were observed starting at laser fluences of 0.1-0.5 J/cm(2) with complete bacteria disintegration at 2-3 J/cm(2) at both wavelengths. Furthermore, ethanol in reaction mixtures significantly (more than one order) enhanced bubble formation phenomena. CONCLUSION: This first application of laser-activated CNTs as PT contrast antimicrobial agents demonstrated its great potential to cause irreparable damages to disease-causing pathogens as well as to detect the pathogens at single bacterium level. This unique integration of laser and nanotechnology may also be used for drinking water treatment, food processing, disinfection of medical instrumentation, and purification of grafts and implants. Furthermore, the significant ethanol-induced enhancement of bubble formation provides another unique possibility to improve the efficiency of selective nanophotothermolysis for treating cancers, wounds, and vascular legions.

In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents.

A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 10(7) normal blood cells.