Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).     

Circulation times of hepatocellular carcinoma cells by in vivo flow cytometry

Hepatocellular carcinoma （HCC） may metastasize to many organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. We have developed the ＂in vivo microscopy＂ to study the mechanisms that govern liver tumor cells spreading through the microenvironment in vivo. A recently developed ＂in vivo flow cytometer＂ combined with real-time confocal fluorescence imaging is used to assess spreading and the circulation kinetics of liver tumor cells. We measure the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which are from the same origin and obtained by repetitive screenings in mice. More than 60% of the HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, less than 40% of the HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.     

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.     

Phase imaging flow cytometry using a focus-stack collecting microscope

This letter introduces a fluidics-based focus-stack collecting microscope. A microfluidic device transports cells through the focal plane of a microscope, resulting in an efficient method to collect focus stacks of large collections of single cells. Images from the focus stacks are used to reconstruct the quantitative phase of cells with the transport-of-intensity-equation method. Using the phase imaging flow cytometer, we measure three-dimensional shape variations of red blood and leukemia cells.     

First characterization of marine particles by laser scanning flow cytometry

A new laser scanning flow cytometer (CLASS) has been developed at ENEA to characterize single marine particles. The first setup of the instrument allowing the detection of the particle light scattering over a wide angular range is presented. A test of CLASS employing polystyrene microspheres is described. Eventually, the first measurements of biological particles (Penicillium Italicum conidia) and marine phytoplankton (Synechocystis cells) are reported.     

Morphometric model of lymphocyte as applied to scanning flow cytometry

The peripheral blood lymphocytes of normal individuals are investigated by methods of specialized light microscopy. Lymphocytes as a whole and T-cell subpopulation are considered. Lymphocyte structure is characterized with reference to polarizing scanning flow cytometry. The lymphocyte and lymphocyte nucleus shapes are analyzed. Linear correlation dependence between sizes of lymphocyte and its nucleus is indicated. A morphometric model of a lymphocyte is constructed using the obtained data. The findings can be used, for instance, as input parameters to solve the direct and inverse light-scattering problems of turbidimetry, nephelometry, and flow cytometry.     

Two-color, double-slit in vivo flow cytometer

The in vivo flow cytometer enables the real-time detection and quantification of fluorescent cells circulating within a live animal without the need for incisions or extraction of blood. It has been used in demonstrating flow velocity disparities in biological flows, and in the investigation of the circulation kinetics of various types of cells. However, a shortcoming of this in vivo flow cytometer is that it provides only one excitation slit at one wavelength, resulting in several performance limitations. Therefore, a second in vivo flow cytometer that provides two different laser wavelengths, 473 and 633 nm, and one or two excitation slits has been designed and built. Thus far, the two-color system has been used to acquire circulation kinetics data of two different cell populations each labeled with a different marker, one cell population labeled with two different markers, and one cell population expressing the green-fluorescent protein gene. In addition, accurate arterial red blood cell velocities within a mouse have been determined using the cytometer.     

Feature-oriented fuzzy bidirectional flow for image enhancement

A fuzzy bidirectional flow is presented, which performs a fuzzy backward (inverse) diffusion along the gradient direction to the isophote lines (edges), while does a certain forward diffusion along the tangent direction on the contrary. Controlled by the image gradient magnitude, the fuzzy membership function guarantees image textures with a natural transition between two different areas. To preserve image features, the nonlinear diffusion coefficients are locally adjusted according to the directional derivatives of the image.     

Characterization of spherical particles using high-order neural networks and scanning flow cytometry

We retrieve the radius R, real n and imaginary k parts of the refractive index of homogeneous spherical particles using angular distribution of the light-scattering intensity. To solve the inverse light-scattering problem we use a high-order neural-network technique. The effect of network parameters on optimization is examined. The technique is evaluated for noise-corrupted input data at 0.6 μm<R<10.6 μm, 1.02<n<1.38, and 0<k<0.03. The errors of retrieval for nonabsorbing particles do not exceed 0.05 μm for radius and 0.015 for refractive index. The experimental verification is fulfilled by experimental data retrieved by means of a scanning flow cytometer. The light-scattering profiles of polystyrene beads and spherized red blood cells are processed with the high-order neural networks and a non-linear regression at Mie theory. The parameters retrieved by the high-order neural networks correlate well with the parameters retrieved by the least-square method.     

光热膨胀材料的光谱特性分析及微型光热驱动机构研究

光热膨胀材料的光谱特性是影响微型光热驱动机构性能的关键因素,该文在理论分析的基础上,采用单积分球测量法对四种聚合物材料的反射光谱及吸收特性进行了实验测量,据此选择制作光热驱动机构的最佳光热膨胀材料,发现掺杂染料的高密度聚乙烯(HDPE)材料在600～690 nm的范围内有较强烈的吸收.选取HDPE作为光热膨胀材料,设计制作了一种长度1 500 μm的微型光热驱动机构,并采用自行研制的CCD显微监控和视频运动测量系统进行了驱动机构的驱动性能测量实验.研究结果表明,在10 mW/650 nm的半导体激光器驱动下,该微型光热驱动机构可以产生18.7 μm的光热偏转量,说明其具有良好的光谱吸收和光热转换特性,可望在微光机电系统(MOEMS)中获得广泛应用.     

Analysis of cell surface molecular distributions and cellular signaling by flow cytometry

Flow cytometry is a fast analysis and separation method for large cell populations, based on collection and processing of optical signals gained on a cell-by-cell basis. These optical signals are scattered light and fluorescence. Owing to its unique potential ofStatistical data analysis and sensitive monitoring of (micro)heterogeneities in large cell populations, flow cytometry—in combination with microscopic imaging techniques—is a powerful tool to study molecular details of cellular signal transduction processes as well. The method also has a widespread clinical application, mostly in analysis of lymphocyte subpopulations for diagnostic (or research) purposes in diseases related to the immune system. A special application of flow cytometry is the mapping of molecular interactions (proximity relationships between membrane proteins) at the cell surface, on a cell-by-cell basis. We developed two approaches to study such questions; both are based ondistance-dependent quenching of excited state fluorophores (donors) by fluorescent or dark (nitroxide radical) acceptors via Förstertype dipole-dipole resonance energy transfer (FRET) and long-range electron transfer (LRET) mechanisms, respectively. A critical evaluation of these methods using donor- or acceptor-conjugated monoclonal antibodies (or their Fab fragments) to select the appropriate cell surface receptor or antigen will be presented in comparison with other approaches for similar purposes. The applicability of FRET and LRET for two-dimensional antigen mapping as well as for detection of conformational changes in extracellular domains of membrane-bound proteins is discussed and illustrated by examples of several lymphoma cell lines. Another special application area of flow cytometry is the analysis of different aspects of cellular signal transduction, e.g., changes of intracellular ion (Ca²⁺, H⁺, Na⁺) concentrations, regulation of ion channel activities, or more complex physiological responses of cell to external stimuli via correlated fluorescence and scatter signal analysis, on a cell-by-cell basis. This way different signaling events such as changes in membrane permeability, membrane potential, cell size and shape, ion distribution, cell density, chromatin structure, etc., can be easily and quickly monitored over large cell populations with the advantage of revealing microheterogeneities in the cellular responses. Flow cytometry also offers the possibility to follow the kinetics of slow (minute- and hour-scale) biological processes in cell populations. These applications are illustrated by the example of complex flow cytometric analysis of signaling in extracellular ATP-triggered apoptosis (programmed cell death) of murine thymic lymphocytes.     

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.     

A study of light scattering of mononuclear blood cells with scanning flow cytometry

This study describes the measurement of light scattering of human mononuclear blood cells, the development of an appropriate optical model for those cells, and solution of the inverse light-scattering problem. The angular dependency of light-scattering intensity of mononuclear blood cells was experimentally measured by means of scanning flow cytometry. A sphere consisting of several concentric homogeneous layers with different refractive indices was tested as an optical model for mononuclear blood cells. A five-layer model has given the best agreement between experimental and theoretical light-scattering profiles. The inverse light-scattering problem was solved for a five-layer model with an optimization procedure that allows one to retrieve cell parameters: cell size relates to the outer diameter of the fifth layer; size of the nucleus relates to the outer diameter of the third layer. Mean values of cell size, nuclear size, refractive indices of nucleus and cellular cytoplasm were determined for blood monocytes and lymphocytes.     

Photothermal waves in a periodically modulated structure

The generation of thermal waves in a one-dimensional structure with a sinusoidal variation in thermal diffusivity can be described by an inhomogeneous Mathieu equation. An exact solution to the frequency-domain Mathieu equation can be found by using a variation of parameters method to give the alternating component of the temperature along the structure. From the theory of Mathieu functions a dispersion relation for the thermal waves is found, which does not display band gaps, but which shows damping that departs from that for thermal waves in a uniform medium.     

Photothermal monitoring of curing in multilayered systems

In this work the process of a curing layer buried inside two external layers is monitored in real time using photothermal radiometry. The curing materials studied are two adhesives and a UV curing resin. It is shown that the curing process can be observed in all the cases as an increase in the thermal diffusion coefficient of the buried sample. It is shown that the kinetic behavior of the adhe- sives is very similar, probably due to the fact that evaporation is the main mechanism that generates the curing contrary to what happens in the UV curing resin where the light flux is the determining factor that induces the polymerization.     

Simultaneous in situ profiling of DNA lesion endpoints based on image cytometry and a single cell database approach

Analyzing the integrity of DNA is one of the most frequent used endpoints for risk assessment of chemical and physical agents. In the framework of a radiobiological space experiment, this work aimed at having (1) a histochemical tool for the in situ assessment of DNA damage in as long as 20 days old fixed cell cultures, (2) a comprehensive tool for the quantification of different types of DNA lesions, and (3) a methodology of sampling thousands of nuclei based on confocal microscopy, automated stage scanning and digital image processing. For this purpose several fixatives and permeabilization techniques were tested together with the combinatorial use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) and the DNA polymerase I mediated in situ nick translation. These biochemical tools are useful for scoring DNA single and double breaks, and oxidative lesions. Ltk(-) cells were exposed either to hydrogen peroxide or heavy ion beam irradiation. Combination of paraformaldehyde fixation, sodium citrate permeabilization and heat gave the best staining results. A three-channel fluorescence methodology was established including a DNA counter stain for nucleus identification and normalization of DNA content. Communication between confocal imaging software, image analysis software and a relational database proved to be pivotal for a semi-automated high-end single cell analysis and storage of images. In this way, DNA damage data per nucleus can be traced back to the original image. As much as 2500 cells could be analyzed in situ within a day and correlations drawn between different DNA lesion endpoints.     

Photothermal radiometry with spherical solids

We extend the applications of photothermal diagnostics to solid spheres, in which both theoretical and experimental PTR studies on spherical geometries and thermal diffusivity of the sample are discussed. Based on the Green function method, a full thermal-wave field distribution of a spherical solid is obtained. Experimental results with steel spheres of different diameters exhibit good agreement between the theory and the experiments.     

Photothermal transmission imaging and microscopy

Examples of photothermal transmission imaging and microscopy are presented. Contrast enhancement in halftone display is achieved using local beam modulation.     

脉冲激光光热失调技术

将脉冲激光作为加热光引入光热失调测量技术中,提出了脉冲激光光热失调测量技术,介绍了其基本原理,分析了脉冲激光光热失调测量技术用于测量光学薄膜微弱吸收的可行性.实验以532 nm的高反射光学薄膜为样品,采用波长为632.8 nm的探测光,研究了脉冲激光光热失调技术信号振幅的时间特性以及最大信号幅值与样品表面加热光能量密度、加热光斑与探测光斑相对位置的变化关系.研究结果表明,光热信号随时间先增大后减小,而随表面加热光能量密度的提高而增大,当样品表面加热和探测光斑重合时光热信号最大.     

In vivo monitoring of multiple circulating cell populations using two-photon flow cytometry

To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.