Integrated photothermal flow cytometry in vivo

The capability of integrated flow cytometry to detect, in real time, moving cells in their natural states in vivo is demonstrated in a study of circulating red and white blood cells in lymph and blood flow of rat mesentery. This system combines dual pump-probe photothermal (PT) techniques, such as PT imaging, the PT thermolens method, and PT velocimetry, with high-resolution (up to 0.3 microm), high-speed (up to 1000 fps) transmission digital microscopy (TDM) and fluorescence imaging. All PT techniques are based on irradiation of cells in rat mesenteric microvessels with a spectrally tunable laser pulse (420 to 570 nm, 8 ns, 0.1 to 300 microJ) and on detection of temperature-dependent variations of the refractive index with a second continuous probe laser beam (633 nm, 1.4 mW). We focus on intravital monitoring of the integral PT response from single, moving, unlabeled cells (from 100 to 500 cells in one measurement). Potential in vivo applications of this new optical tool, called PT flow cytometry (PTFC), are discussed, including identification of selected cells with differences in natural absorptive properties and sizes, determination of laser-induced cell damage, estimation of flow velocity, and monitoring of circulating cells labeled with PT probes.     

Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

Red blood cells (RBCs) have been found to undergo "programmed cell death," or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.     

A novel photoacoustic tomography based on a time-resolved technique and an acoustic lens imaging system

A novel photoacoustic (PA) tomographic method, which is based on a time-resolved technique and an acoustic lens imaging system, is presented in this paper. A YAG laser operating at 532 nm with a 7 ns pulse width and 10 mJ optical pulse is employed as the excitation source to irradiate the tissue. PA signals generated from the tissue are imaged onto a multi-element linear array transducer with an acoustic lens. A 64 electronic switch is efficiently used for changing the parallel PA signals into a series. The proposed method directly provides PA images without any complex reconstruction algorithms. With the time-resolved technique, tomographic imaging can be achieved successfully. The results show that the images agree well with the original samples.     

Superhigh-sensitivity photothermal monitoring of individual cell response to antitumor drug

We describe and explore the capability of a photothermal (PT) assay with modified schematics for highly sensitive detection of individual cell response to antitumor drug impact in vitro. Specifically, we used the nonlinear differential PT test to measure distinctive changes of specific PT parameters after exposure of KB3 carcinoma cells to the antitumor drug vinblastine in the broad concentration range of 10(-10) to 300 nM. Verification of the PT assay was performed by comparison with multidrug-resistant cells and comparison with conventional assays evaluating cell viability, cytochrome c release, apoptosis induction, and cell size. We demonstrate that this system is capable of detecting drug-induced signals at a concentration threshold sensitivity at least seven orders of magnitude better than existing assays. We anticipate that this technique may serve as a convenient and rapid analytical tool to evaluate the presence of intracellular drug, with applications in high throughput screening assays and for studying drug uptake and distribution in more complex biological or clinical samples.     

Photothermal responses of individual cells

Photothermal (PT) responses of individual intact cells are studied with a thermal lens dual-laser scheme. A multiparameter model for analysis of PT responses as a function of cell size, structure, and optical properties is suggested and verified experimentally for living cells, red blood cells, lymphocytes, tumor cells (K 562), hepatocytes, and miocytes, by applying pulsed laser radiation at 532 nm for 10-ns duration. PT responses for noninvasive and damaging modes of laser-cell interaction are investigated. It is shown theoretically and experimentally that specific optical and structural features of cells influence the polarity, shape, front, and tail lengths of their PT responses. Common for different cells, features of PT responses are evaluated. It is found that in cells with a highly heterogeneous light-absorbing structure, the PT response of a whole cell differs from that of the local absorbing area. The model suggested allows us to interpret PT responses from single cells and to compare cells in terms of their diameter, degree of spatial heterogeneity of light absorbance, and laser-induced damage thresholds.     

Near-infrared transmittance pulse oximetry with laser diodes

Pulse oximeters are widely used for noninvasive monitoring of oxygen saturation in arterial blood hemoglobin. We present a transmittance pulse oximetry system based on near-infrared (NIR) laser diodes (750 and 850 nm) for monitoring oxygen saturation of arterial blood hemoglobin. The pulse oximetry system is made up of the optical sensor, sensor electronics, and processing block. Also, we show experimental results obtained during the development of the whole NIR transmittance pulse oximetry system along with modifications in the sensor configuration, signal processing algorithm, and calibration procedure. Issues concerning wavelength selection and its implications for the improvement of the transmittance pulse oximetry technique are discussed. The results obtained demonstrate the proposed system's usefulness in monitoring a wide range of oxygen saturation levels.     

Improving drug accumulation and photothermal efficacy in tumor depending on size of ICG loaded lipid-polymer nanoparticles

A key challenge to strengthen anti-tumor efficacy is to improve drug accumulation in tumors through size control. To explore the biodistribution and tumor accumulation of nanoparticles, we developed indocyanine green (ICG) loaded poly (lactic-co-glycolic acid) (PLGA) -lecithin-polyethylene glycol (PEG) core-shell nanoparticles (INPs) with 39 nm, 68 nm and 116 nm via single-step nanoprecipitation. These INPs exhibited good monodispersity, excellent fluorescence and size stability, and enhanced temperature response after laser irradiation. Through cell uptake and photothermal efficiency in vitro, we demonstrated that 39 nm INPs were more easily be absorbed by pancreatic carcinoma tumor cells (BxPC-3) and showed better photothermal damage than that of 68 nm and 116 nm size of INPs. Simultaneously, the fluorescence of INPs offered a real-time imaging monitor for subcellular locating and in vivo metabolic distribution. Near-infrared imaging in vivo and photothermal therapy illustrated that 68 nm INPs showed the strongest efficiency to suppress tumor growth due to abundant accumulation in BxPC-3 xenograft tumor model. The findings revealed that a nontoxic, size-dependent, theranostic INPs model was built for in vivo cancer imaging and photothermal therapy without adverse effect.     

Cell disintegration by laser-induced transient microbubbles and its simultaneous monitoring by interferometry

Selective retina treatment (SRT) is a novel short pulsed laser therapy of several retinal diseases associated with a decreased metabolism at the retinal pigment epithelium (RPE). The range of laser pulse energies is small, in which the desired selective RPE disintegration is achieved without adverse effects to the neural retina. Thus, a real-time dosimetry control is required. We investigated a noninvasive interferometric technique able to monitor microbubble formation around the intracellular melanin granula, which is the origin of the desired RPE damage. A porcine ex vivo RPE model was irradiated by single pulses (350 ns1.7 mus) of a neodymium: yttrium lithium fluoride laser (527 nm). The specimen was simultaneously probed by a Michelson interferometer (helium neon-laser: 633 nm) and by a hydrophone. Cell viability assays (Calcein-AM) were performed after irradiation. At threshold radiant exposure for cell death (ED(50)=129+/-5 mJ cm2 for 350 ns; ED50=180+/-5 mJ cm2 for 1.7 mus), the interferometric transients changed due to microbubble formation. No major differences in the bubble dynamics were observed between both pulse durations. An algorithm to determine cell death from the interferometric transients showed less than 10% false positive or false negative results for the applied laser expositions compared to the viability assay. Interferometry is a reliable noncontact technique to monitor RPE disintegration and may serve as real-time dosimetry control during SRT.     

Spectroscopic investigations of carious tooth decay

We report on the elemental composition of healthy and infected part of human tooth using laser induced breakdown spectroscopy (LIBS). We have used prominent constituent transitions in laser-excited tooth to diagnose the state of the tooth. A nanosecond laser pulse (355nm, 5ns) was used as an ablating pulse and the sodium (3s2S-3p2P) at 588.99 and (3s2S-3p2P) at 589.99nm, strontium (5s21S-1s5P) at 460.55nm, and calcium (3d3D-4f 3F0) at 452.55nm transitions for spectroscopic analysis. The spectroscopic observations in conjunction with discriminate analysis showed that calcium attached to the hydroxyapatite structure of the tooth was affected severely at the infected part of the tooth. The position-time plots generated from two-dimensional (2D) images conclusively showed a decrease in calcium concentration in the infected region of the irradiated tooth. Using the technique, we could distinguish between the healthy and carious parts of the tooth with significant accuracy.     

Optimal ultraviolet wavelength for in vivo photoacoustic imaging of cell nuclei

In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.     

Raman microspectroscopy and imaging provides insights into heme aggregation and denaturation within human erythrocytes

The oxygenation process of a human erythrocyte is monitored using a Raman microimaging technique. Raman images of the 1638 cm(-1) band are recorded in the oxygenated and deoxygenated state using only 120 s of laser exposure and approximately 1 mW of defocused laser power. The images show hemoglobin oxygenating and deoxygenating within the cell. Prolonged laser imaging exposure (<180 s) at low temperatures results in photoinduced and/or thermal degradation. The effect of thermal degradation is investigated by recording spectra of erythrocytes as a function of temperature between 4 and 52 degrees C. Five bands at 1396, 1365, 1248, 972, and 662 cm(-1) are identified as markers for heme aggregation. Raman images recorded of cells after prolonged laser exposure appear to show heme aggregation commencing in the middle and moving toward the periphery of the cell. UV-visible spectra of erythrocytes show the Soret band to be broader and red shifted (approximately 3 nm) at temperatures between 45 and 55 degrees indicative of excitonic interactions. It is postulated that the enhancement of the aggregation marker bands observed at 632.8-nm excitation results primarily from excitonic interactions between the aggregated hemes in response to protein denaturation. The results have important medical implications in detecting and monitoring heme aggregation associated with hemopathies such as sickle cell disease.     

Optoacoustic determination of optical attenuation depth using interferometric detection

We use a modified Mach-Zehnder interferometer to measure surface displacement resulting from the thermoelastic response of a target to the absorption of a short laser pulse with axial and temporal resolutions of 0.1 nm and 3 ns, respectively. These measurements are used in conjunction with a solution to the thermoelastic wave equation and a nonlinear optimization algorithm to extract optical attenuation depth. We demonstrate the ability to determine the effective optical attenuation depth of homogeneous targets with either diffuse or specular reflecting surfaces with a precision of 相似文献   

Depth profiling of photothermal compound concentrations using phase sensitive optical coherence tomography

A model that describes the concentration of photothermal (light-to-heat converters) compounds as a function of depth in a turbid medium is developed. The system consists of a pump laser (808 nm modulated at 400 Hz), which heats a photothermal compound, and a phase sensitive spectral domain optical coherence tomography system, which detects the changes in the optical path length of the sample induced by the temperature increase. The model is theoretically derived and the coefficients are empirically determined using solid homogeneous gel phantoms. The model is validated by reconstructing the concentration of a photothermal compound in thick single and double layer solid phantoms.     

Coherent thermal wave imaging of subsurface chromophores in biological materials

Thermal wave imaging of discrete subsurface chromophores in biological materials is reported using a phase sensitive coherent detection technique applied to recorded infrared (IR) images. We demonstrate that utilization of a periodically modulated laser source for thermal wave excitation and coherent detection applied to each pixel may be used to compute images of thermal wave amplitude and phase at the laser modulation frequency. In comparison to recorded IR images, the narrow-band detection technique significantly improves the quality of thermal wave amplitude images of subsurface chromophores in biological materials. Additionally, the technique provides phase information, which may be used to estimate chromophore depth in tissue. Application of the technique is demonstrated using tissue phantoms and in vivo biological models. We present a theoretical analysis and computer simulations that demonstrate the effect of tissue optical and thermal properties on thermal wave amplitude and phase. In comparison to the pulsed photothermal technique, coherent thermal wave imaging of subsurface chromophores in tissue for diagnostic applications allows reduction of peak incident laser fluence by as much as four orders of magnitude and is safer and more amenable to in vivo imaging.     

纳米硫化铜载药红细胞靶向蓄积及提高肿瘤光热治疗效果研究

光热治疗偶联剂纳米硫化铜的临床应用,存在增加浓度、提高光热治疗效果的同时也增加毒副作用的矛盾.利用红细胞作为纳米硫化铜载体,体外激光照射控制其在肿瘤组织释放,增强光热治疗效果.采用改良低渗预膨胀法,将纳米硫化铜包载入红细胞,构建纳米硫化铜载药红细胞(CuS@ER);用小动物活体成像表征其肿瘤靶向效率,评价光热升温效果....     

Target-specific near-IR induced drug release and photothermal therapy with accumulated Au/Ag hollow nanoshells on pulmonary cancer cell membranes

Au/Ag hollow nanoshells (AuHNSs) were developed as multifunctional therapeutic agents for effective, targeted, photothermally induced drug delivery under near-infrared (NIR) light. AuHNSs were synthesized by galvanic replacement reaction. We further conjugated antibodies against the epidermal growth factor receptor (EGFR) to the PEGylated AuHNS, followed by loading with the antitumor drug doxorubicin (AuHNS-EGFR-DOX) for lung cancer treatment. AuHNSs showed similar photothermal efficiency to gold nanorods under optimized NIR laser power. The targeting of AuHNS-EGFR-DOX was confirmed by light-scattering images of A549 cells, and doxorubicin release from the AuHNSs was evaluated under low pH and NIR-irradiated conditions. Multifunctional AuHNS-EGFR-DOX induced photothermal ablation of the targeted lung cancer cells and rapid doxorubicin release following irradiation with NIR laser. Furthermore, we evaluated the effectiveness of AuHNS-EGFR-DOX drug delivery by comparing two drug delivery methods: receptor-mediated endocytosis and cell-surface targeting. Accumulation of the AuHNS-EGFR-DOX on the cell surfaces by targeting EGFR turned out to be more effective for lung cancer treatments than uptake of AuHNS-EGFR-DOX. Taken together, our data suggest a new and optimal method of NIR-induced drug release via the accumulation of targeted AuHNS-EGFR-DOX on cancer cell membranes.     

Nanocluster model of photothermal assay: application for high-sensitive monitoring of nicotine-induced changes in metabolism, apoptosis, and necrosis at a cellular level

This study evaluates the capability of a photothermal (PT) assay to monitor the impact of nicotine on pancreatic cancer cells (AR42J). The specific PT response is closely proportional to nicotine concentrations at concentration range 1 nM to 100 microM, while at high concentrations of nicotine ranging from 1 mM to 50 mM, PT response shows dramatic decrease. According to the theoretical model, the mechanism of the PT assay is associated with metabolic and apoptotic-related shrinking of local cellular absorbing nanoscale zones caused by increased local absorption at low nicotine doses, while high doses of nicotine lead to apoptotic release of absorbing component (cytochrome c) into the intracellular space, and necrotic swelling of organelles, thereby causing a decrease in local absorption. This model is verified with conventional imaging and with Annexin-V Propidium iodide kits. The PT assay, in addition to its high sensitivity (3 orders of magnitude better than conventional assay), shows the potential to distinguish between various functional states of cells that are associated with changes in metabolism, early and late stages of apoptosis, and necrosis. Comparison of PT responses of pancreatic tumor cells AR42J with isolated primary pancreatic acinar cells and HepG2 cells shows a universal nature of PT assay.     

基于时域后向投影的光声谱成像系统研究

光声谱成像是一种新的生物组织成像方法,它结合光学成像和超声成像的特点,可提供高分辨率和对比度的图像。采用波长532nm、重复频率10Hz的脉冲激光作为激励源,宽带PVDF非聚焦超声探测器频率响应范围为200kHz～15MHz,探测器以圆周扫描的方式采集样品的时域光声信号,并采用时域后向投影算法重建样品内部的二维光学吸收分布图像。实验表明,系统成像的空间分辨率小于1mm,重建的图像与原始样品完全吻合。     

Continuous-wave infrared optical nerve stimulation for potential diagnostic applications

Optical nerve stimulation using infrared laser radiation has recently been developed as a potential alternative to electrical nerve stimulation. However, recent studies have focused primarily on pulsed delivery of the laser radiation and at relatively low pulse rates. The objective of this study is to demonstrate faster optical stimulation of the prostate cavernous nerves using continuous-wave (cw) infrared laser radiation for potential diagnostic applications. A thulium fiber laser (λ=1870 nm) is used for noncontact optical stimulation of the rat prostate cavernous nerves in vivo. Optical nerve stimulation, as measured by an intracavernous pressure (ICP) response in the penis, is achieved with the laser operating in either cw mode, or with a 5-ms pulse duration at 10, 20, 30, 40, 50, and 100 Hz. Successful optical stimulation is observed to be primarily dependent on a threshold nerve temperature (42 to 45 °C), rather than an incident fluence, as previously reported. cw optical nerve stimulation provides a significantly faster ICP response time using a lower power (and also less expensive) laser than pulsed stimulation. cw optical nerve stimulation may therefore represent an alternative mode of stimulation for intraoperative diagnostic applications where a rapid response is critical, such as identification of the cavernous nerves during prostate cancer surgery.     

Magnetic-field-assisted photothermal therapy of cancer cells using Fe-doped carbon nanoparticles

Photothermal therapy with assistance of nanoparticles offers a solution for the destruction of cancer cells without significant collateral damage to otherwise healthy cells. However, minimizing the required number of injected nanoparticles is a major challenge. Here, we introduce the use of magnetic carbon nanoparticles (MCNPs), localizing them in a desired region by applying an external magnetic-field, and irradiating the targeted cancer cells with a near-infrared laser beam. The MCNPs were prepared in benzene, using an electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements showed the size distribution of these MCNPs to be in the range of 5 to 10 nm. For photothermal irradiation, a tunable continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely guide photothermal therapy.