KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01-3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT: ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

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.

Real-time monitoring of circulating tumor cell (CTC) release after nanodrug or tumor radiotherapy using in vivo flow cytometry.

Noninvasive biological readouts of tumor metastatic risk and therapeutic efficacy are needed as healthcare costs rise. CTCs are the source of metastasis in distant organs that are responsible for the majority of cancer-related deaths. Here we demonstrate the acute and long-term effect of vascular disrupting therapies (high-dose radiotherapy and tumor necrosis factor-alpha (TNF)) on CTCs released from the primary tumor with a non-invasive real-time in vivo flow cytometry system. Using our innovative flow cytometry platform, we show here that radiation and nanodrug treatment can lead to short term release of CTC from the primary tumor. There was no increase in metastasis frequency or extent between control and TNF-treated mice; however, a significant reduction in lung metastasis was noted in the radiotherapy alone group. Mice treated with both TNF and radiotherapy had a slightly elevated metastatic profile between that of radiation alone and control (untreated) tumors. Possible mechanisms based on therapy specific vessel disruption and cell death are discussed. Overall, CTCs correlated with tumor progression and suggest CTC enumeration described herein may be useful in clinical management of solid tumor malignancies.

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.

Towards rainbow portable Cytophone with laser diodes for global disease diagnostics.

In vivo, Cytophone has demonstrated the capability for the early diagnosis of cancer, infection, and cardiovascular disorders through photoacoustic detection of circulating disease markers directly in the bloodstream with an unprecedented 1,000-fold improvement in sensitivity. Nevertheless, a Cytophone with higher specificity and portability is urgently needed. Here, we introduce a novel Cytophone platform that integrates a miniature multispectral laser diode array, time-color coding, and high-speed time-resolved signal processing. Using two-color (808 nm/915 nm) laser diodes, we demonstrated spectral identification of white and red clots, melanoma cells, and hemozoin in malaria-infected erythrocytes against a blood background and artifacts. Data from a Plasmodium yoelii murine model and cultured human P. falciparum were verified in vitro with confocal photothermal and fluorescent microscopy. With these techniques, we detected infected cells within 4 h after invasion, which makes hemozoin promising as a spectrally selective marker at the earliest stages of malaria progression. Along with the findings from our previous application of Cytophone with conventional lasers for the diagnosis of melanoma, bacteremia, sickle anemia, thrombosis, stroke, and abnormal hemoglobin forms, this current finding suggests the potential for the development of a portable rainbow Cytophone with multispectral laser diodes for the identification of these and other diseases.

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 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.

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.

Bioinspired magnetic nanoparticles as multimodal photoacoustic, photothermal and photomechanical contrast agents.

Nanoparticles from magnetotactic bacteria have been used in conventional imaging, drug delivery, and magnetic manipulations. Here, we show that these natural nanoparticles and their bioinspired hybrids with near-infrared gold nanorods and folic acid can serve as molecular high-contrast photoacoustic probes for single-cell diagnostics and as photothermal agents for single-cell therapy using laser-induced vapor nanobubbles and magnetic field as significant signal and therapy amplifiers. These theranostics agents enable the detection and photomechanical killing of triple negative breast cancer cells that are resistant to conventional chemotherapy, with just one or a few low-energy laser pulses. In studies in vivo, we discovered that circulating tumor cells labeled with the nanohybrids generate transient ultrasharp photoacoustic resonances directly in the bloodstream as the basis for new super-resolution photoacoustic flow cytometry in vivo. These properties make natural and bioinspired magnetic nanoparticles promising biocompatible, multimodal, high-contrast, and clinically relevant cellular probes for many in vitro and in vivo biomedical applications.

Photoacoustic and fluorescent effects in multilayer plasmon-dye interfaces.

Progress in understanding the cell biology and diseases depends on advanced imaging and labeling techniques. Here, we address this demand by exploring novel multilayered nanocomposites (MNCs) with plasmonic nanoparticles and absorbing dyes in thin nonabsorbing shells as supercontrast multimodal photoacoustic (PA) and fluorescent agents in the near-infrared range. The proof of concept was performed with gold nanorods (GNRs) and indocyanine green (ICG) dispersed in a matrix of biodegradable polymers. We demonstrated synergetic PA effects in MNCs with the gold-ICG interface that could not be achieved with ICG and GNRs alone. We also observed ultrasharp PA and emission peaks that could be associated with nonlinear PA and spaser effects, respectively. Low-toxicity multimodal MNCs with unique plasmonic, thermal and acoustic properties have the potential to make a breakthrough in PA flow cytometry and near-infrared spasers in vivo by using the synergetic interaction of plasmonic modes with a nearby absorbing medium.

New Frontiers in Diagnosis and Therapy of Circulating Tumor Markers in Cerebrospinal Fluid In Vitro and In Vivo.

One of the greatest challenges in neuro-oncology is diagnosis and therapy (theranostics) of leptomeningeal metastasis (LM), brain metastasis (BM) and brain tumors (BT), which are associated with poor prognosis in patients. Retrospective analyses suggest that cerebrospinal fluid (CSF) is one of the promising diagnostic targets because CSF passes through central nervous system, harvests tumor-related markers from brain tissue and, then, delivers them into peripheral parts of the human body where CSF can be sampled using minimally invasive and routine clinical procedure. However, limited sensitivity of the established clinical diagnostic cytology in vitro and MRI in vivo together with minimal therapeutic options do not provide patient care at early, potentially treatable, stages of LM, BM and BT. Novel technologies are in demand. This review outlines the advantages, limitations and clinical utility of emerging liquid biopsy in vitro and photoacoustic flow cytometry (PAFC) in vivo for assessment of CSF markers including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), microRNA (miRNA), proteins, exosomes and emboli. The integration of in vitro and in vivo methods, PAFC-guided theranostics of single CTCs and targeted drug delivery are discussed as future perspectives.

In vivo liquid biopsy using Cytophone platform for photoacoustic detection of circulating tumor cells in patients with melanoma.

Most cancer deaths arise from metastases as a result of circulating tumor cells (CTCs) spreading from the primary tumor to vital organs. Despite progress in cancer prognosis, the role of CTCs in early disease diagnosis is unclear because of the low sensitivity of CTC assays. We demonstrate the high sensitivity of the Cytophone technology using an in vivo photoacoustic flow cytometry platform with a high pulse rate laser and focused ultrasound transducers for label-free detection of melanin-bearing CTCs in patients with melanoma. The transcutaneous delivery of laser pulses via intact skin to a blood vessel results in the generation of acoustic waves from CTCs, which are amplified by vapor nanobubbles around intrinsic melanin nanoclusters. The time-resolved detection of acoustic waves using fast signal processing algorithms makes photoacoustic data tolerant to skin pigmentation and motion. No CTC-associated signals within established thresholds were identified in 19 healthy volunteers, but 27 of 28 patients with melanoma displayed signals consistent with single, clustered, and likely rolling CTCs. The detection limit ranged down to 1 CTC/liter of blood, which is ~1000 times better than in preexisting assays. The Cytophone could detect individual CTCs at a concentration of ≥1 CTC/ml in 20 s and could also identify clots and CTC-clot emboli. The in vivo results were verified with six ex vivo methods. These data suggest the potential of in vivo blood testing with the Cytophone for early melanoma screening, assessment of disease recurrence, and monitoring of the physical destruction of CTCs through real-time CTC counting.

Dynamic blood flow phantom with negative and positive photoacoustic contrasts.

In vivo photoacoustic (PA) flow cytometry (PAFC) has great clinical potential for early, noninvasive diagnosis of cancer, infections (e.g., malaria and bacteremia), sickle anemia, and cardiovascular disorders, including stroke prevention through detection of circulating white clots with negative PA contrast. For clinical applications, this diagnostic platform still requires optimization and calibration. We have already demonstrated that this need can be partially addressed by in vivo examination of large mouse blood vessels, which are similar to human vessels used. Here, we present an alternative method for PAFC optimization that utilizes novel, clinically relevant phantoms resembling pigmented skin, tissue, vessels, and flowing blood. This phantom consists of a scattering-absorbing medium with a melanin layer and plastic tube with flowing beads to model light-absorbing red blood cells (RBCs) and circulating tumor cells (CTCs), as well as transparent beads to model white blood cells and clots. Using a laser diode, we demonstrated the extraordinary ability of PAFC to dynamically detect fast-moving mimic CTCs with positive PA contrast and white clots with negative PA contrast in an RBC background. Time-resolved detection of the delayed PA signals from blood vessels demonstrated complete suppression of the PA background from the modeled pigmented skin. This novel, medically relevant, dynamic blood flow phantom can be used to calibrate and maintain PAFC parameters for routine clinical applications.

Noninvasive label-free detection of circulating white and red blood clots in deep vessels with a focused photoacoustic probe.

Blood clotting is a serious clinical complication of many medical procedures and disorders including surgery, catheterization, transplantation, extracorporeal circuits, infections, and cancer. This complication leads to high patient morbidity and mortality due to clot-induced pulmonary embolism, stroke, and in some cases heart attack. Despite the clear medical significance, little progress has been made in developing the methods for detection of circulating blood clots (CBCs), also called emboli. We recently demonstrated the application of in vivo photoacoustic (PA) flow cytometry (PAFC) with unfocused ultrasound transducers for detection of CBCs in small vessels in a mouse model. In the current study, we extend applicability of PAFC for detection of CBCs in relatively large (1.5-2 mm) and deep (up to 5-6 mm) blood vessels in rat and rabbit models using a high pulse rate 1064 nm laser and focused ultrasound transducer with a central hole for an optic fiber. Employing phantoms and chemical activation of clotting, we demonstrated PA identification of white, red, and mixed CBCs producing negative, positive, and mixed PA contrast in blood background, respectively. We confirmed that PAFC can detect both red and white CBCs induced by microsurgical procedures, such as a needle or catheter insertion, as well as stroke modeled by injection of artificial clots. Our results show great potential for a PAFC diagnostic platform with a wearable PA fiber probe for diagnosis of thrombosis and embolism in vivo that is impossible with existing techniques.

High-speed microscopy for in vivo monitoring of lymph dynamics.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-µm microspheres as cell phantoms on slides and in 90-µm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Photoacoustic in vitro flow cytometry for nanomaterial research.

Conventional flow cytometry is a versatile tool for drug research and cell characterization. However, it is poorly suited for quantification of non-fluorescent proteins and artificial nanomaterials without the use of additional labeling. The rapid growth of biomedical applications for small non-fluorescent nanoparticles (NPs) for drug delivery and contrast and therapy enhancement, as well as research focused on natural cell pigments and chromophores, demands high-throughput quantification methods for the non-fluorescent components. In this work, we present a novel photoacoustic (PA) fluorescence flow cytometry (PAFFC) platform that combines NP quantification though PA detection with conventional in vitro flow cytometry sample characterization using fluorescence labeling. PAFFC simplifies high-throughput analysis of cell-NP interactions, optimization of targeted nanodrugs, and NP toxicity assessment, providing a direct correlation between NP uptake and characterization of toxicity markers for every cell.

Spaser as a biological probe.

Understanding cell biology greatly benefits from the development of advanced diagnostic probes. Here we introduce a 22-nm spaser (plasmonic nanolaser) with the ability to serve as a super-bright, water-soluble, biocompatible probe capable of generating stimulated emission directly inside living cells and animal tissues. We have demonstrated a lasing regime associated with the formation of a dynamic vapour nanobubble around the spaser that leads to giant spasing with emission intensity and spectral width >100 times brighter and 30-fold narrower, respectively, than for quantum dots. The absorption losses in the spaser enhance its multifunctionality, allowing for nanobubble-amplified photothermal and photoacoustic imaging and therapy. Furthermore, the silica spaser surface has been covalently functionalized with folic acid for molecular targeting of cancer cells. All these properties make a nanobubble spaser a promising multimodal, super-contrast, ultrafast cellular probe with a single-pulse nanosecond excitation for a variety of in vitro and in vivo biomedical applications.

Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo.

Control of sickle cell disease (SCD) stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic (PA) flow cytometry (PAFC) has a potential for real-time monitoring of circulating sickled cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT) and PA spectral imaging of sickle red blood cells (sRBCs) expressing SCD-associated hemoglobin (HbS) compared to normal red blood cells (nRBCs). We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2-4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial hemoglobin heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode) as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs both in vitro and in vivo.