Lymph Liquid Biopsy for Detection of Cancer Stem Cells.

Collection of a blood sample defined by the term "blood liquid biopsy" is commonly used to detect diagnostic, prognostic, and therapeutic decision-making markers of metastatic tumors including circulating tumor cells (CTCs). Many tumors also release CTCs and other markers into lymph fluid, but the utility of lymphatic markers largely remains unexplored. Here, we introduce lymph liquid biopsy through collection of peripheral (afferent) and central (thoracic duct [TD]) lymph samples and demonstrates its feasibility for detection of stem-like CTCs potentially responsible for metastasis development and tumor relapse. Stemness of lymphatic CTCs (L-CTCs) was determined by spheroid-forming assay in vitro. Simultaneously, we tested blood CTCs by conventional blood liquid biopsy, and monitored the primary tumor size, early metastasis in a sentinel lymph node (SLN) and distant metastasis in lungs. Using a mouse model at early melanoma stage with no distant metastasis, we identified stem-like L-CTCs in lymph samples from afferent lymphatic vessels. Since these vessels transport cells from the primary tumor to SLN, our finding emphasizes the significance of the lymphatic pathway in development of SLN metastasis. Surprisingly, in pre-metastatic disease, stem-like L-CTCs were detected in lymph samples from the TD, which directly empties lymph into blood circulation. This suggests a new contribution of the lymphatic system to initiation of distant metastasis. Integration of lymph and blood liquid biopsies demonstrated that all mice with early melanoma had stem-like CTCs in at least one of three samples (afferent lymph, TD lymph, and blood). At the stage of distant metastasis, spheroid-forming L-CTCs were detected in TD lymph, but not in afferent lymph. Altogether, our results demonstrated that lymph liquid biopsy and testing L-CTCs holds promise for diagnosis and prognosis of early metastasis. © 2020 International Society for Advancement of Cytometry.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry.

Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multicolor in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed. © 2018 International Society for Advancement of Cytometry.

In vivo photoacoustic flow cytometry for early malaria diagnosis.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.

Photothermal confocal multicolor microscopy of nanoparticles and nanodrugs in live cells.

Growing biomedical applications of non-fluorescent nanoparticles (NPs) for molecular imaging, disease diagnosis, drug delivery, and theranostics require new tools for real-time detection of nanomaterials, drug nano-carriers, and NP-drug conjugates (nanodrugs) in complex biological environments without additional labeling. Photothermal (PT) microscopy (PTM) has enormous potential for absorption-based identification and quantification of non-fluorescent molecules and NPs at a single molecule and 1.4 nm gold NP level. Recently, we have developed confocal PTM providing three-dimensional (3D) mapping and spectral identification of multiple chromophores and fluorophores in live cells. Here, we summarize recent advances in the application of confocal multicolor PTM for 3D visualization of single and clustered NPs, alone and in individual cells. In particular, we demonstrate identification of functionalized magnetic and gold-silver NPs, as well as graphene and carbon nanotubes in cancer cells and among blood cells. The potential to use PTM for super-resolution imaging (down to 50 nm), real-time NP tracking, guidance of PT nanotherapy, and multiplex cancer markers targeting, as well as analysis of non-linear PT phenomena and amplification of nanodrug efficacy through NP clustering and nano-bubble formation are also discussed.

Super-resolution nonlinear photothermal microscopy.

Super-resolution fluorescence microscopy enables imaging of fluorescent structures beyond the diffraction limit. However, this technique cannot be applied to weakly fluorescent cellular components or labels. As an alternative, photothermal microscopy based on nonradiative transformation of absorbed energy into heat has demonstrated imaging of nonfluorescent structures including single molecules and ~1-nm gold nanoparticles. However, previously photothermal imaging has been performed with a diffraction-limited resolution only. Herein, super-resolution, far-field photothermal microscopy based on nonlinear signal dependence on the laser energy is introduced. Among various nonlinear phenomena, including absorption saturation, multiphoton absorption, and signal temperature dependence, signal amplification by laser-induced nanobubbles around overheated nano-objects is explored. A Gaussian laser beam profile is used to demonstrate the image spatial sharpening for calibrated 260-nm metal strips, resolving of a plasmonic nanoassembly, visualization of 10-nm gold nanoparticles in graphene, and hemoglobin nanoclusters in live erythrocytes with resolution down to 50 nm. These nonlinear phenomena can be used for 3D imaging with improved lateral and axial resolution in most photothermal methods, including photoacoustic microscopy.

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.

Nanotheranostics of circulating tumor cells, infections and other pathological features in vivo.

Many life-threatening diseases are disseminated through biological fluids, such as blood, lymph, and cerebrospinal fluid. The migration of tumor cells through the vascular circulation is a mandatory step in metastasis, which is responsible for ∼90% of cancer-associated mortality. Circulating pathogenic bacteria, viruses, or blood clots lead to other serious conditions including bacteremia, sepsis, viremia, infarction, and stroke. Therefore, technologies capable of detecting circulating tumor cells (CTCs), circulating bacterial cells (CBCs), circulating endothelial cells (CECs), circulating blood clots, cancer biomarkers such as microparticles and exosomes, which contain important microRNA signatures, and other abnormal features such as malaria parasites in biological fluids may facilitate early diagnosis and treatment of metastatic cancers, infections, and adverse cardiovascular events. Unfortunately, even in a disease setting, circulating abnormal cells are rare events that are easily obscured by the overwhelming background material in whole blood. Existing detection methods mostly rely on ex vivo analyses of limited volumes (a few milliliters) of blood samples. These small volumes limit the probability of detecting CTCs, CECs, CBCs and other rare phenomena. In vivo detection platforms capable of continuously monitoring the entire blood volume may substantially increase the probability of detecting circulating abnormal cells and, in particular, increase the opportunity to identify exceedingly rare and potentially dangerous subsets of these cells, such as circulating cancer stem cells (CCSCs). In addition, in vivo detection technologies capable of destroying and/or capturing circulating abnormal cells may inhibit disease progression. This review focuses on novel therapeutic and diagnostic (theranostic) platforms integrating in vivo real-time early diagnosis and nano-bubble based targeted therapy of CTCs, CECs, CBCs and other abnormal objects in circulation. This critical review particularly focuses on nanotechnology-based theranostic (nanotheranostic) approaches, especially in vivo photoacoustic (PA) and photothermal (PT) nanotheranostic platforms. We emphasize an urgent need for in vivo platforms composed of multifunctional contrast nanoagents, which utilize diverse modalities to realize a breakthrough for early detection and treatment of harmful diseases disseminated through the circulation.

Photoacoustic flow cytometry.

Conventional flow cytometry using scattering and fluorescent detection methods has been a fundamental tool of biological discoveries for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents the long-term study of cells in their native environment. Here, we summarize recent advances of new generation flow cytometry for in vivo noninvasive label-free or targeted detection of cells in blood, lymph, bone, cerebral and plant vasculatures using photoacoustic (PA) detection techniques, multispectral high-pulse-repetition-rate lasers, tunable ultrasharp (up to 0.8 nm) rainbow plasmonic nanoprobes, positive and negative PA contrasts, in vivo magnetic enrichment, time-of-flight cell velocity measurement, PA spectral analysis, and integration of PA, photothermal (PT), fluorescent, and Raman methods. Unique applications of this tool are reviewed with a focus on ultrasensitive detection of normal blood cells at different functional states (e.g., apoptotic and necrotic) and rare abnormal cells including circulating tumor cells (CTCs), cancer stem cells, pathogens, clots, sickle cells as well as pharmokinetics of nanoparticles, dyes, microbubbles and drug nanocarriers. Using this tool we discovered that palpation, biopsy, or surgery can enhance CTC release from primary tumors, increasing the risk of metastasis. The novel fluctuation flow cytometry provided the opportunity for the dynamic study of blood rheology including red blood cell aggregation and clot formation in different medical conditions (e.g., blood disorders, cancer, or surgery). Theranostics, as a combination of PA diagnosis and PT nanobubble-amplified multiplex therapy, was used for eradication of CTCs, purging of infected blood, and thrombolysis of clots using PA guidance to control therapy efficiency. In vivo flow cytometry using a portable fiber-based devices can provide a breakthrough platform for early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack by well-timed personalized therapy.

Photothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores.

Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.

In vivo photoacoustic and photothermal cytometry for monitoring multiple blood rheology parameters.

Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo assessment of multiple hemorheological parameters over long periods of time has not been reported. Here, we review the capabilities of label-free photoacoustic (PA) and photothermal (PT) flow cytometry for dynamic monitoring of hemorhelogical parameters in vivo which we refer to as photoacoustic and photothermal blood rheology. Using phenomenological models, we analyze correlations between both PT and PA signal characteristics in the dynamic modes and following determinants of blood rheology: red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, hematocrit, and likely shear rate. We present ex vivo and in vivo experimental verifications involving high-speed PT imaging of RBCs, identification of sickle cells in a mouse model of human sickle cell disease and in vivo monitoring of complex hemorheological changes (e.g., RBC deformability, hematocrit and RBC aggregation). The multi-parameter platform that integrates PT, PA, and conventional optical techniques has potential for translation to clinical applications using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency.

In vivo ultra-fast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers.

The circulating tumor cells (CTCs) appear to be a marker of metastasis development, especially, for highly aggressive and epidemically growing melanoma malignancy that is often metastatic at early stages. Recently, we introduced in vivo photoacoustic (PA) flow cytometry (PAFC) for label-free detection of mouse B16F10 CTCs in melanoma-bearing mice using melanin as an intrinsic marker. Here, we significantly improve the speed of PAFC by using a high-pulse repetition rate laser operating at 820 and 1064 nm wavelengths. This platform was used in preclinical studies for label-free PA detection of low-pigmented human CTCs. Demonstrated label-free PAFC detection, low level of background signals, and favorable safety standards for near-infrared irradiation suggest that a fiber laser operating at 1064 nm at pulse repetition rates up to 0.5 MHz could be a promising source for portable clinical PAFC devices. The possible applications can include early diagnosis of melanoma at the parallel progression of primary tumor and CTCs, detection of cancer recurrence, residual disease and real-time monitoring of therapy efficiency by counting CTCs before, during, and after therapeutic intervention. Herewith, we also address sensitivity of label-free detection of melanoma CTCs and introduce in vivo CTC targeting by magnetic nanoparticles conjugated with specific antibody and magnetic cells enrichment.

In vivo plant flow cytometry: a first proof-of-concept.

In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugates uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature.

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume.

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 µm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted.

Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.

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.

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.

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.

In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease.

The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.