In vivo flow cytometer for real-time detection and quantification of circulating cells

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.     

Retinal flow cytometer

The in vivo flow cytometer is an instrument capable of continuous, real-time monitoring of fluorescently labeled cells in the circulation without the need to draw blood samples. However, the original system probes a single vessel in the mouse ear; the small sample volume limits the sensitivity of the technique. We describe an in vivo retinal flow cytometer that simultaneously probes five artery-vein pairs in the mouse eye by circularly scanning a small laser spot rapidly around the optic nerve head. We demonstrate that the retinal flow cytometer detects about five times more cells per minute than the original in vivo flow cytometer does in the ear.     

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 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.     

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.     

Measurement Conditions for Flow Cytometry Analyses of Cell Lines from Urological Carcinomas

Prerequisites for successful flow cytometry investigations are specific antibodies labeled with appropriate fluorochromes and negligible autofluorescence of the untreated cells at the wavelength of interest. The aim of this study was (a) to characterize frequently used urological carcinoma cell lines with regard to their autofluorescence properties, (b) to demonstrate the autofluorescence as a serious interfering factor on FACS analysis of urological carcinoma cell lines and (c) to suggest an alternative to avoid interfering autofluorescence. Twenty-one cell lines originating from prostate carcinoma, renal cell carcinoma and bladder cancer were included in this study. The various cell lines were read on a flow cytometer in comparison to human erythrocytes as cells with low fluorescence intensity. Urological cell lines show a high autofluorescence when flow cytometry analyses are performed at the frequently used excitation wavelengths at 405 and 488 nm. At excitation wavelength of 633 nm, this problem was reduced and most of the cell lines (14/21) were without autofluorescence at the emission wavelength of 785 nm. In addition, with a spectrofluorometer three exemplary cell lysates were investigated. The above observations were confirmed. The dye APC-Cy7 is one suitable fluorochrome for successful investigation under these measurement conditions.     

Development of a single‐cell X‐ray fluorescence flow cytometer

An X‐ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X‐ray beam. X‐ray fluorescence was then used to determine the mass of metal in each cell. By making single‐cell measurements, the population heterogeneity for metals in the µM to mM concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approach has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ～4 cells min^?1. These data show evidence for surprisingly broad metal distributions. Details of the device design, data analysis and opportunities for further sensitivity improvement are described.     

Quantitative evaluation of radiation dose by γ-H2AX on a microfluidic chip in a miniature fluorescence cytometer

Evaluation of radiation dose is very important for the detection of radiation damage. γ-H2AX is a popular biological dosimeter to evaluate the radiation effect. Typically, bulky and expensive commercial flow cytometers are used to detect γ-H2AX. This paper presents a miniaturized and high sensitive cytometer using a microfluidic chip for evaluating the radiation dose by detecting the mean immunofluorescence intensity of γ-H2AX. A compact optical focusing system and a shift-phase differential amplifier are designed to improve the detection sensitivity. Sample lymphocyte cells are stained by FITC fluorescent dye after being irradiated by UVC. Comparison experiments between the developed miniature cytometer and a commercial flow cytometer were conducted under different radiation doses. The developed microfluidic cytometer also demonstrates a good linear correlation between the measured fluorescence intensity and the irradiation dose with a detection limit similar to that of the commercial flow cytometer. The developed cytometer can evaluate quantitatively the radiation dose by the mean fluorescence intensity of γ-H2AX with a significantly smaller amount of blood samples than a commercial flow cytometer.     

The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope,Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells

The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.     

人肝癌SMMC-7721细胞对低剂量γ射线辐射超敏感性的研究

研究了人肝癌细胞SMMC-7721在低剂量γ射线照射下超敏感性和增强的辐射抗性响应。选用对数生长期细胞接受0—6 Gy不同剂量的^60Coγ射线的照射。利用流式细胞仪对细胞进行分选计数,并用克隆形成法检测细胞存活率。发现SMMC-7721细胞存在低剂量辐射超敏感性和增强的辐射抗性响应,即在0—0．3Gy之间细胞表现出单位剂量杀伤增强现象,在0．3—1Gy细胞表现一定的辐射抗性,在1Gy以上,细胞的存活符合线性平方模型。     

First studies of pico- and nanoplankton populations by a laser scanning flow cytometer

The innovative laser scanning flow cytometer (LSFC) CLASS, developed at ENEA Research Center of Frascati, merges the ability to perform very fast analyses on a statistically significant number of particles to the capability of characterizing, from a morphological and optical point of view, cells even if they are not fluorescent. In this work two cultured populations of picoplankton and nanoplankton have been analyzed by means of CLASS to evaluate the future applicability of this system as a tool for the early detection of algal blooms. The analyses were performed on a red pigmented Synechococcus sp. and Chlamydomonas reinhardtii by measuring simultaneously the side scattering, scattering in the wide angular range 5-100°, fluorescence in two spectral bands and depolarization of the laser light by every single cell contained in the sample. The results highlighted the ability of CLASS to characterize cell populations in which standard flow cytometers encounter difficulties and in which the use of traditional time-consuming image analyses is often required, such as those characterized by different morphologies in a small size with little or no fluorescence. In particular the system presented was demonstrated to be suitable for the study of composition and changes in a phytoplankton population.     

Determination of vessel cross section for flow rate quantification

This study was motivated by the interest of measuring different cardiac parameters for which changes in the flow rate during a cardiac cycle needs to be determined at different positions along a vessel segment. These measurements result in a great number of images for which automatic contour detection is very helpful. A model-based algorithm for intraluminal contour detection has been developed in order to allow an accurate quantitative image analysis. The algorithm permits to select contours automatically on all the frames and slices of an imaging study. Images obtained on a flow phantom simulating the effects of blood circulation in large arteries have been used to validate the method. They were acquired with a specially designed interleaved multi slice and phase sequence, using a standard whole-body 2 Tesla NMR scanner. A potential in vivo application of the algorithm has been demonstrated on abdominal aorta images.     

A bioreactor for in-cell protein NMR

The inside of the cell is a complex environment that is difficult to simulate when studying proteins and other molecules in vitro. We have developed a device and system that provides a controlled environment for nuclear magnetic resonance (NMR) experiments involving living cells. Our device comprises two main parts, an NMR detection region and a circulation system. The flow of medium from the bottom of the device pushes alginate encapsulated cells into the circulation chamber. In the chamber, the exchange of oxygen and nutrients occurs between the media and the encapsulated cells. When the media flow is stopped, the encapsulated cells fall back into the NMR detection region, and spectra can be acquired. We have utilized the bioreactor to study the expression of the natively disordered protein α-synuclein, inside Escherichia coli cells.     

Discriminating Multiplexed GFP Reporters in Primary Articular Chondrocyte Cultures Using Image Cytometry

Flow cytometry has become a standard tool for defining a heterogeneous cell population based on surface expressed epitopes or GFP reporters that reflect cell types or cellular differentiation. The introduction of image cytometry raised the possibility of adaptation to discriminate GFP reporters used to appreciate cell heterogeneity within the skeletal lineages. The optical filters and LEDs were optimized for the reporters used in transgenic mice expressing various fluorescent proteins. In addition, the need for compensation between eGFP and surrounding reporters due to optical cross-talk was eliminated by selecting the appropriate excitation and emission filters. Bone marrow or articular cartilage cell cultures from GFP and RFP reporter mouse lines were established to demonstrate the equivalency in functionalities of image to flow cytometry analysis. To examine the ability for monitoring primary cell differentiation, articular chondrocyte cell cultures were established from mice that were single or doubly transgenic (Dkk3eGFP and Col2A1GFPcyan), which identify the progression of superficial small articular cell to a mature chondrocyte. The instrument was able to rapidly and accurately discriminate cells that were Dkk3eGFP only, Dkk3eGFP/Col2A1GFPcyan, and Col2A1GFP, which provides a useful tool for studying the impact of culture conditions on lineage expansion and differentiation.     

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).     

In vivo MR spectroscopy and MR imaging on non-anaesthetized marine fish: techniques and first results

In vivo Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) experiments were carried out in non-anaesthetized marine fish with a maximum body length of 40 cm using a 4.7 T horizontal magnet. In flow-through animal chambers long-term investigations could be carried out for several days in a sessile benthic zoarcid species, the eelpout Zoarces viviparus, as well as in a pelagic gadid species, the cod Gadus morhua. Sea water adapted probes and optimised excitation and refocusing pulse shapes reduced the negative effects of the highly conductive medium. In both species, in vivo 31P-NMR spectra were collected within 5 minutes with a reasonable signal to noise ratio (PCr/Pi ratio > 40 in cod) and typical line widths in the range of 20 to max. 40 Hz. Magnetic resonance images were recorded in the sessile species without movement artefacts. In these fish it was even possible to measure the blood flow of different vessels with a flow compensated gradient echo sequence and to carry out localized in vivo 1H-NMR spectroscopy in different organs.     

In vivo photoacoustic time-of-flight velocity measurement of single cells and nanoparticles

Optical techniques for in vivo measurement of blood flow velocity are not quite applicable for determination of velocity of individual cells or nanoparticles. Here, we describe a photoacoustic time-of-flight method to measure the velocity of individual absorbing objects by using single and multiple laser beams. Its capability was demonstrated in vitro on blood vessel phantom and in vivo on an animal (mouse) model for estimating velocity of gold nanorods, melanin nanoparticles, erythrocytes, leukocytes, and circulating tumor cells in the broad range of flow velocity from 0.1?mm/s to 14?cm/s. Object velocity can be used to identify single cells circulating at different velocities or cell aggregates and to determine a cell's location in a vessel cross-section.     

Photobleaching with a subnanosecond laser flash

In standard fluorescence recovery after photobleaching (FRAP) applications for measuring lateral diffusion rates and adsorption/desorption kinetics of fluorescent molecules at biological or model membranes, irreversible bleaching is induced by a bright excitation flash of at least millisecond time scale. It has been presumed that the bleaching event is of a low probability and the significant bleached population that develops during the flash results from each molecule undergoing thousands of excitation/deexcitation cycles before a bleaching event occurs. In some FRAP experiments, notably polarized FRAP (PFRAP) for measuring molecular rotational diffusion rates, it is desirable to use much shorter (subnanosecond) bleaching pulses. However, subnanosecond pulses are shorter than the fluorescence lifetime, so that any fluorophore will experience at most only one visit to the excited state during the bleaching pulse. If bleaching occurs only by the same processes as in slower FRAP experiments, one would thereby expect only minimal bleaching regardless of the bleach intensity. Moreover, the ability of fast polarized pulses to imprint an anisotropic orientational pattern in the postbleach unbleached fluorophore, an ability essential for PFRAP, is not at all guaranteed, particularly if two-photon processes are involved in high-intensity short bleach pulses. In this study, bleaching depths are measured as a function of subnanosecond pulse intensity on a small labeled protein covalently immobilized on fused silica. We show that bright subnanosecond laser flashes do indeed produce significant bleaching, that both two photon effects and reversible bleaching are involved, and that polarized bleaching does produce an anisotropic orientational pattern of unbleached fluorophore. We also postulate a theoretical molecular state model which semiquantitatively accounts for the experimentally observed dependence of reversible bleaching on bleaching pulse intensity.     

Cerebral blood flow imaged with ultrahigh-resolution optical coherence angiography and Doppler tomography

Speckle contrast based optical coherence angiography (OCA) and optical coherence Doppler tomography (ODT) have been applied to image cerebral blood flow previously. However, the contrast mechanisms of these two methods are not fully studied. Here, we present both flow phantom and in vivo animal experiments using ultrahigh-resolution OCA (μOCA) and ODT (μODT) to investigate the flow sensitivity differences between these two methods. Our results show that the high sensitivity of μOCA for visualizing minute vasculature (e.g., slow capillary beds) is due to the enhancement by random Brownian motion of scatterers (e.g., red and white blood cells) within the vessels; whereas, μODT permits detection of directional flow below the Brownian motion regime (e.g., laser-induced microischemia) and is, therefore, more suitable for brain functional imaging.