Instrument for fluorescence sensing of circulating cells with diffuse light in mice in vivo

Accurate quantification of circulating cell populations in mice is important in many areas of preclinical biomedical research. Normally, this is done either by extraction and analysis of small blood samples or, more recently, by using microscopy-based in vivo fluorescence flow cytometry. We describe a new technological approach to this problem using detection of diffuse fluorescent light from relatively large blood vessels in vivo. The diffuse fluorescence flow cytometer (DFFC) uses a laser to illuminate a mouse limb and an array of optical fibers coupled to a high-sensitivity photomultiplier tube array operating in photon counting mode to detect weak fluorescence signals from cells. We first demonstrate that the DFFC instrument is capable of detecting fluorescent microspheres and Vybrant-DiD-labeled cells in a custom-made optical flow phantom with similar size, optical properties, linear flow rates, and autofluorescence as a mouse limb. We also present preliminary data demonstrating that the DFFC is capable of detecting circulating cells in nude mice in vivo. In principle, this device would allow interrogation of the whole blood volume of a mouse in minutes, with sensitivity improvement by several orders of magnitude compared to current approaches.     

Removal of Out-of-Plane Fluorescence for Single Cell Visualization and Quantification in Cryo-Imaging

We developed a cryo-imaging system, which alternates between sectioning (10–40 μm) and imaging bright field and fluorescence block-face image volumes with micron-scale-resolution. For applications requiring single-cell detection of fluorescently labeled cells anywhere in a mouse, we are developing software for reduction of out-of-plane fluorescence. In mouse experiments, we imaged GFP-labeled cancer and stem cells, and cell-sized fluorescent microspheres. To remove out-of-plane fluorescence, we used a simplified model of light-tissue interaction whereby the next-image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing an objective function on subtracted images. Tissue-specific attenuation parameters [μ _T: heart (267 ± 47.6 cm⁻¹), liver (218 ± 27.1 cm⁻¹), brain (161 ± 27.4 cm⁻¹)] were found to be within the range of estimates in the literature. “Next-image” processing removed out-of-plane fluorescence equally well across multiple tissues (brain, kidney, liver, etc.), and analysis of 200 microsphere images gave 97 ± 2% reduction of out-of-plane fluorescence. Next-image processing greatly improved axial-resolution, enabled high quality 3D volume renderings, and improved automated enumeration of single cells by up to 24%. The method has been used to identify metastatic cancer sites, determine homing of stem cells to injury sites, and show microsphere distribution correlated with blood flow patterns.     

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ～500?μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30?μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8?cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.     

Multi-dimensional laser confocal microscopy on live cells in submicroliter volumes using glass capillaries

Imaging live cells using laser confocal microscopy requires the use of complex and rather cumbersome incubation chamber systems in order to maintain the correct physiological conditions. The volume of these chambers is in the range of a few hundred microliters. Here we present an easy and convenient alternative in the form of glass capillaries that accommodate volumes of 0.2-10 microliters. The capillaries can be loaded with both suspension and adherent cells. The loaded capillaries are taped on microscope slides and submerged into the immersion oil that covers the objective. The correct temperature is maintained using a thermostat-controlled objective heater. We demonstrate that using microlens enhanced -rotating Nipkow disc based confocal illumination, in combination with cold CCD cameras, maximum resolution multicolor time lapse fluorescence images can be obtained from live cells. The images obtained are free from disturbing optical distortions. Imaging in submicroliter volumes allows for fluorescence visualization of very rare cell types isolated using flow or affinity sorting or obtained by fine needle biopsies.     

Colon cancer cells in peripheral blood of cancerous tamarins

Diagnosing colon cancer in its early stages would lower the mortality rate. The cotton-top tamarin, Saguinus oedipus, serves as a model for the study of human colon cancer. This New World monkey has a high incidence of colitis and colon cancer. The mouse anti-human monoclonal antibody BR55.2, with specificity for human colon adenocarcinoma, was biotinylated. Peripheral blood mononuclear cells (PBMC) from animals with colon cancer were fluorescently stained with the biotinylated BR55.2. These results showed the cross-reactivity of mouse anti-human colon cancer monoclonal antibody to the PBMC of cancerous tamarins. Antibodies from either cancerous or chronic colitis tamarins were also biotinylated. Fluorescently labeled cells were detected when PBMC from cancerous tamarins were incubated with biotinylated antibodies from cancerous tamarins. Cytofluorographic analysis also showed a significant 4.5-fold difference in the percentage of fluorescently labeled PBMC between cancerous and chronic colitis tamarins when stained with biotinylated antibodies from cancerous tamarins. DNA flow cytometry analysis showed that PBMC from cancerous tamarins have a higher percentage of aneuploid cells than PBMC from chronic colitis tamarins.     

Investigations of the eye fundus using a simultaneous optical coherence tomography/indocyanine green fluorescence imaging system

We develop a dual-channel optical coherence tomography/indocyanine green (OCT/ICG) fluorescence system based on our previously reported ophthalmic OCT/confocal imaging system. The confocal channel is tuned to the fluorescence wavelength range of the ICG dye and light from the same optical source is used to generate the OCT image and to excite the ICG fluorescence. The system enables the clinician to visualize simultaneously en face OCT slices and corresponding ICG angiograms of the ocular fundus, displayed side by side. C-scan (constant depth) and B-scan (cross section) images are collected by fast en face scanning (T-scan). The pixel-to-pixel correspondence between the OCT and angiography images enables the user to precisely capture OCT B-scans at selected points on the ICG confocal images.     

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.     

Fiber optic probe cytometer

An optical fiber probe is used to both excite and collect fluorescence from a suspension of cells. The configuration of the probe is such that one or a few cells are sensed at a time, with a convenient cell concentration. With fluorescently labeled antibodies to cellular antigens, the fiber optic cytometer is able to identify the presence of a specific set of cells with high sensitivity.     

Spatial Distribution of Platelet Deposition in Stented Arterial Models Under Physiologic Flow

This paper presents dynamic flow experiments with fluorescently labeled platelets to allow for spatial observation of wall attachment in inter-strut spacings, to investigate their relationship to flow patterns. Human blood with fluorescently labeled platelets was circulated through an in vitro system that produced physiologic pulsatile flow in a parallel plate flow chamber that contained three different stent designs that feature completely recirculating flow, partially recirculating flow (intermediate strut spacing), and completely reattached flow. Highly resolved spatial distribution of platelets was obtained by imaging fluorescently labeled platelets between the struts. Platelet deposition was higher in areas where flow is directed towards the wall, and lower in areas where flow is directed away from the wall. Flow detachment and reattachment points exhibited very low platelet deposition. Platelet deposition within intermediate strut spacing continued to increase throughout the experimental period, indicating that the deposition rate had not plateaued unlike other strut spacings. The spatial uniformity and temporal increase in platelet deposition for the intermediate strut spacing confirms and helps explain our previous finding that platelet deposition was highest with this strut spacing. Further experimental investigations will include more complex three-dimensional geometries.     

热变性对血细胞流式细胞术分析结果的影响

目的 初步探讨热变性对外周血细胞在流式细胞术分析结果中的影响,从而提高流式-荧光原位杂交（Flow-FISH）技术的应用.方法 收集5例非骨髓造血干细胞疾病患者肝素抗凝外周血标本,以CD45-Alexa Fluor（R）647标记细胞表面抗原,高温变性后,采用流式细胞术分析热变性前后外周血有核细胞的散射光信号和荧光信号变化.结果 热变性后,外周血粒细胞的侧向散射光明显缩小;单核细胞不易通过散射光被区分和设门.所有细胞CD45表达强度均减弱,以淋巴细胞为著;通过侧向散射光和CD45设门虽能大致区分各群细胞,但不及未热变性细胞清晰.结论 热变性后,外周血细胞在流式细胞术中的散射光信号和荧光信号均发生了变化,按照常规FSC/SSC和CD45/SSC方法设门进行细胞亚群分析的结果不精确,利用系列特异性荧光抗体标记是一种有前景的方法.     

Leukocyte compartments in the mouse lung: distinguishing between marginated, interstitial, and alveolar cells in response to injury

We developed a flow cytometry-based assay to simultaneously quantify multiple leukocyte populations in the marginated vascular, interstitial, and alveolar compartments of the mouse lung. An intravenous injection of a fluorescently labeled anti-CD45 antibody was used to label circulating and marginated vascular leukocytes. Following vascular flushing to remove non-adherent cells and collection of broncho-alveolar lavage (BAL) fluid, lungs were digested and a second fluorescent anti-CD45 antibody was added ex vivo to identify cells not located in the vascular space. In the na?ve mouse lung, we found about 11 million CD45+ leukocytes, of which 87% (9.5 million) were in the vascular marginated compartment, consisting of 17% NK cells, 17% neutrophils, 57% mononuclear myeloid cells (monocytes, macrophage precursors and dendritic cells), and 10% T cells (CD4+, CD8+, and invariant NKT cells). Non-vascular compartments including the interstitial compartment contained 7.7×10(5)cells, consisting of 49% NK cells, 25% dendritic cells, and 16% other mononuclear myeloid cells. The alveolar compartment was overwhelmingly populated by macrophages (5.63×10(5)cells, or 93%). We next studied leukocyte margination and extravasation into the lung following acid injury, a model of gastric aspiration. At 1 h after injury, neutrophils were markedly elevated in the blood while all other circulating leukocytes declined by an average of 79%. At 4 h after injury, there was a peak in the numbers of marginated neutrophils, NK cells, CD4+ and CD8+ T cells and a peak in the number of alveolar NK cells. Most interstitial cells consisted of DCs, neutrophils, and CD4+ T cells, and most alveolar compartment cells consisted of macrophages, neutrophils, and NK cells. At 24 h after injury, there was a decline in the number of all marginated and interstitial leukocytes and a peak in alveolar neutrophils. In sum, we have developed a novel assay to study leukocyte margination and trafficking following pulmonary inflammation and show that marginated cells comprise a large fraction of lung leukocytes that increases shortly after lung injury. This assay may be of interest in future studies to determine if leukocytes become activated upon adherence to the endothelium, and have properties that distinguish them from interstitial and circulating cells.     

Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption flow cytometric analysis.

Evaluation of blood cells to determine immunologic status is becoming an important clinical application of flow cytometric analysis. For a wider use of immunophenotyping technology in clinical laboratories, the authors developed a rapid method to detect monoclonal antibody-labeled cells using forward light scatter/absorption clinical flow cytometers such as the Technicon H*1 and Technicon H*2 differential complete blood count analyzers. Calf-intestinal alkaline phosphatase was conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer at pH 9.6 was selected as buffer/substrate to yield stable, insoluble, and very intense purplish-blue precipitates on the surface of the cells labeled with monoclonal antibody-alkaline phosphatase conjugates. Endogenous alkaline phosphatase in granulocytes was inhibited with levamisole. Early mild fixation of the white cells permitted incubation at 38 +/- 1 degrees C, which accelerated each step of the reaction without disrupting the cells throughout the procedure. The method is competitive with the direct immunofluorescence whole-blood method used on fluorescence flow cytometers in speed, sensitivity, and accuracy, as demonstrated with alkaline phosphatase-conjugated anti-CD2, CD3, CD4, CD8, CD19 monoclonal antibodies.     

Imaging tumor angiogenesis with fluorescent proteins

We have developed three unique mouse models to image angiogenesis with fluorescent proteins, which are described in this review. First, we have adapted the surgical orthotopic implantation (SOI) model to image angiogenesis of human tumors labeled with green fluorescent protein (GFP) transplanted in nude mice. The nonluminous induced capillaries are clearly visible by contrast against the very bright tumor fluorescence examined either intravitally or by whole-body imaging in real time. Intravital images of an SOI model of human pancreatic tumors expressing GFP visualized angiogenic capillaries at both primary and metastatic sites. Whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period in an SOI model of human breast cancer expressing GFP. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold and enabling vessels to be externally visualized in GFP-expressing tumors growing on internal organs. The second model utilizes dual-color fluorescence imaging, effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice that express GFP in all cells. This dual-color model visualizes with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis. The GFP-expressing tumor vasculature, both nascent and mature, are readily distinguished interacting with the RFP-expressing tumor cells. Using a spectral imaging system based on liquid crystal tunable filters, we were able to separate individual spectral species on a pixel-by-pixel basis. Such techniques non-invasively visualized the presence of host GFP-expressing vessels within an RFP-labeled orthotopic human breast tumor by real-time whole-body imaging. The third model involves a transgenic mouse in which the regulatory elements of the stem cell marker nestin drive GFP. The nestin-GFP mouse expresses GFP in areas of the brain, hair follicle stem cells, and in a network of blood vessels in the skin interconnecting hair follicles. RFP-expressing tumors transplanted to nestin-GFP mice enable specific visualization of nascent vessels in skin-growing tumors such as melanoma. Thus, fluorescent proteins expressed in vivo offer very high resolution and sensitivity for real-time imaging of angiogenesis.     

X chromosome territories in human female lymphocytes.

We developed an improved bromodeoxyuridine (BrdU)-DNA assay procedure for flow cytometric analysis. Which makes possible cytochemical investigation against cells after BrdU-DNA assay, such as immunochemical staining or in situ hybridization. Using this method, we performed fluorescence in situ hybridization (FISH) technique using DNA probes that recognize whole X chromosome of human lymphocytes in peripheral blood (resting cells) and cultured cells stimulated by phytohemagglutinin (PHA) (proliferative cells). Cultured cells were fractionated precisely into each stage of cell cycle by cell sorter after flow cytometric analysis. Following FISH procedure, the cells were examined for the volumes of two X chromosome territories in the nuclei using the optical section images on a confocal laser scanning microscope (CLSM). And we analyzed the variation in the volume ratio (R) of two X chromosome territories at different stages in cell cycle. Our data indicated that there were no significant difference between the volume of two X chromosome territories in female human lymphocytes at resting (G0) stage. Furthermore, they also showed no significant alternation throughout the cell cycle. These results may challenge the traditional concepts for inactivated chromosome in two points. First, the inactivated X chromosome is more decondensed throughout the cell cycle than previously thought. Second, although the inactivated X chromosome appears more decondensed during its self replication it dose not bring a great change in the volume of its territory.     

Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa1 or B1 exhibited the strongest labeling whereas OKT4+ cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.     

Portable two-color in vivo flow cytometer for real-time detection of fluorescently-labeled circulating cells

The recent introduction of the in vivo flow cytometer for real-time, noninvasive detection and quantification of cells circulating in the vasculature of small animals has provided a powerful tool for tracking the roles of different types of cells in disease progression. We describe a portable version of the device, which provides the capability to: a) excite and detect fluorescence at two distinct colors simultaneously, and b) perform data analysis in real time. These advances improve significantly the utility of the instrument and provide a means of increasing detection specificity. As examples, we present the depletion kinetics of circulating green fluorescent protein (GFP)-labeled breast cancer cells in the vasculature of mice, and the specific detection of circulating hematopoietic stem cells labeled in vivo with two antibodies.     

Time‐lapse microscopy of macrophages during embryonic vascular development

Background : Macrophages are present before the onset of blood flow, but very little is known about their function in vascular development. We have developed a technique to concurrently label both endothelial cells and macrophages for time‐lapse microscopy using co‐injection of fluorescently conjugated acetylated low‐density lipoprotein (AcLDL) and phagocytic dye PKH26‐PCL. Results : We characterize double‐labeled cells to confirm specific labeling of macrophages. Double‐labeled cells circulate, roll along the endothelium, and extravasate from vessels. Most observed macrophages are integrated into the vessel wall, showing an endothelial‐like morphology. We used transgenic quail that express a fluorescent protein driven by the endothelial‐specific promoter Tie1 in conjugation with the phagocytic dye to analyze these cells. Circulating PKH26‐PCL‐labeled cells are mostly Tie1?, but those which have integrated into the vessel wall are largely Tie1+. The endothelial‐like phagocytic cells were generally stationary during normal vascular development. We, therefore, induced vascular remodeling and found that these cells could be recruited to sites of remodeling. Conclusions : The active interaction of endothelial cells and macrophages support the hypothesis that these cells are involved in vascular remodeling. The presence of phagocytic endothelial‐like cells suggests either a myeloid‐origin to certain endothelial cells or that circulating endothelial cells/hematopoietic stem cells have phagocytic capacity. Developmental Dynamics 241:1423–1431, 2012. © 2012 Wiley Periodicals, Inc.     

基于暗视场荧光图像分析的大鼠微血管流速测量系统的建立

将荧光标记的自身红细胞注入SD大鼠体内,在荧光显微镜下观测标记红细胞在大鼠微血管中的流动情况,并通过显微摄像系统将整个过程以视频信号的形式存贮。使用视频采集卡将流速变化过程回放采样,得到暗视场下的荧光图像。然后利用帧图像分离出奇偶场的图像分析方法测定血流速度。在该系统下测量流动小室中荧光小球的流速,得到的测量值与实际值之间的误差小于7%,两者没明显的差异(P>0.05),流速测量的上限为9.6mm/s。并在大鼠微循环障碍研究中,应用此系统得到了血流速度随时间变化的情况。     

Fast optically sectioned fluorescence HiLo endomicroscopy

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 μm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.     

Confocal fluorescent intravital microscopy of the murine spleen

Intravital microscopy has provided many insights into cellular interactions in various secondary lymphoid tissues. Because this technique allows for the visualization of cellular movement in real-time, it has been very powerful. However, until now, it has been difficult to apply this technique to the spleen. We report a technique that utilizes the Nikon RCM-8000 scanning laser, confocal microscope that allows for visualization of cellular movement in real-time in the rodent spleen. Using fluorescently labeled high molecular weight dextran or monoclonal antibodies, we are able to visualize fluorescently labeled cells rolling, tethering, and adhering in the spleen. In addition, we show that the majority of blood flow to the spleen remains within the white pulp nodules, as do most transferred erythrocytes at early time points. This is the first report of intravital microscopy of the spleen using a method that allows for easy identification of transferred cells.