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).     

Real-time phase-resolved functional optical coherence tomography by use of optical Hilbert transformation

We have developed a novel real-time phase-resolved functional optical coherence tomography system that uses optical Hilbert transformation. When we use a resonant scanner in the reference arm of the interferometer, with an axial scanning speed of 4 kHz, the frame rate of both structural and Doppler blood-flow imaging with a size of 100 by 100 pixels is 10 Hz. The system has high sensitivity and a larger dynamic range for measuring the Doppler frequency shift that is due to moving red blood cells. Real-time images of in vivo blood flow in human skin obtained with this interferometer are presented.     

Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering

Purpose

To examine mesenchymal stem cell (MSC) labeling with micrometer-sized iron oxide particles (MPIOs) for magnetic resonance imaging (MRI)-based tracking and its application to monitoring articular cartilage regeneration.

Methods

Rabbit MSCs were labeled using commercial MPIOs. In vitro MRI was performed with gradient echo (GRE) and spin echo (SE) sequences at 3T and quantitatively characterized using line profile and region of interest analysis. Ex vivo MRI of hydrogel-encapsulated labeled MSCs implanted within a bovine knee was performed with spoiled GRE (SPGR) and T_1ρ sequences. Fluorescence microscopy, labeling efficiency, and chondrogenesis of MPIO-labeled cells were also examined.

Results

MPIO labeling results in efficient contrast uptake and signal loss that can be visualized and quantitatively characterized via MRI. SPGR imaging of implanted cells results in ex vivo detection within native tissue, and T_1ρ imaging is unaffected by the presence of labeled cells immediately following implantation. MPIO labeling does not affect quantitative glycosaminoglycan production during chondrogenesis, but iron aggregation hinders extracellular matrix visualization. This aggregation may result from excess unincorporated particles following labeling and is an issue that necessitates further investigation.

Conclusion

This study demonstrates the promise of MPIO labeling for monitoring cartilage regeneration and highlights its potential in the development of cell-based tissue engineering strategies.     

In vivo assessment of the permeability of the blood--brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish

ABSTRACT: BACKGROUND: Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood--brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB. METHODS: We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7--8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs. RESULTS: The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs. CONCLUSIONS: We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.     

High-resolution in vivo imaging of blood vessels without labeling

We demonstrate that both oxyhemoglobin and deoxyhemoglobin have sequential two-color, two-photon absorption properties that can serve as endogenous contrasts in microvasculature imaging. Using a sensitive modulation transfer technique, we are able to image hemoglobin in red blood cells with micrometer resolution, both in vitro and in vivo. We show that excellent contrast from hemoglobin without any labeling can be obtained in tissue.     

MR tracking of magnetically labeled mesenchymal stem cells in rats with liver fibrosis

Purpose

In vivo magnetic resonance (MR) tracking of magnetically labeled bone marrow mesenchymal stem cells (BMSCs) administered via the mesenteric vein to rats with liver fibrosis.

Materials and Methods

Rat BMSCs were labeled with superparamagnetic iron oxide (SPIO) and the characteristics of the BMSCs after labeling were investigated. Eighteen rats with CCL4-induced liver fibrosis were randomized to three groups to receive SPIO-labeled BMSCs (BMSC-labeled group), cell-free SPIO (SPIO group), or unlabeled BMSCs (control group). MR imaging of the liver was performed at different time points, and signal-to-noise ratio (SNR) of the liver was measured. In vivo distribution of delivered BMSCs was assessed by histological analysis.

Results

Labeling of BMSCs with SPIO did not significantly alter cell viability and proliferation activity. In BMSC-labeled group, the liver SNR immediately decreased from 8.56±0.26 to 3.53±0.41 at 1 h post injection and remained at a significantly lower level till 12 days (P<.05 versus the level before). By contrast, the liver SNR of the SPIO group almost recovered to the preinjection level (P=.125) at 3 days after a transient decrease. In control group, the liver SNR demonstrated no significant difference at the tested time points. Additionally, Prussian blue-positive cells were mainly distributed in the liver parenchyma, especially in injured areas.

Conclusion

The magnetically labeled BMSCs infused through the mesenteric vein can be detected in the fibrotic liver of rats using in vivo MR imaging up to 12 days after injection.     

Corticospinal interaction during isometric compensation for modulated forces with different frequencies

Background

The zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.

Results

We screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1 μg/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.

Conclusions

The coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.     

Optimization of the Near-Infrared Fluorescence Labeling for In Vivo Monitoring of a Protein Drug Distribution in Animal Model

The objective of this study is to optimize the parameters in labeling near-infrared (NIR)fluorescent dye cypate to protein drugs for in vivo optical imaging of drug distributions in animal model. l-ASparaginase (l-ASNase) was used as a protein drug model for the study. To achieve this goal, various labeling conditions, including different catalysts, feed ratios of all components, pH conditions, temperatures, and reacting durations, were investigated. The dye-to-protein (D/P) ratio and enzymatic activity were designated as the metric to evaluate the labeling process. The stability of the cypate–protein conjugate in blood serum and its distribution in small animals were subsequently inspected. Results showed that feed ratio of l-ASNase and the pH value played the most important role in adjusting the labeling efficiency. Reaction duration and temperature had less effect on the dye-to-protein labeling properties. The optimal condition for the labeling of cypate to l-ASNase was 4 h reaction duration at 4 °C and pH 8.5 under catalysis by HOBt/HBTU. The dynamic distribution in animal model displayed that the labeled l-ASNase firstly accumulated in liver and cleared from the enteron system. This study demonstrated that the NIR image system combined with NIR probe has the capability to trace the dynamics of protein drugs in animals for drug development.     

Method for intracellular magnetic labeling of human mononuclear cells using approved iron contrast agents

A method for intracellular iron labeling of human mononuclear cells (lymphocytes and monocytes) for magnetic resonance imaging (MRI) using simple incubation of cells with approved MRI iron contrast agents is presented. Labeled cells can be detected by MRI in vitro, and this suggests the possibility that the technique could become a marker for in vivo lymphocyte and monocyte trafficking studies in acute inflammatory lesions such as those in Multiple Sclerosis.     

Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.     

Tracking of transplanted mesenchymal stem cells labeled with fluorescent magnetic nanoparticle in liver cirrhosis rat model with 3-T MRI

In vivo visualization of transplanted stem cells with noninvasive technique is essential for the monitoring of cell implantation, homing and differentiation. At present, superparamagnetic iron oxide (SPIO) is most commonly used for cell labeling. However, stem cells lack phagocytic capacity and transfection agent is required for sufficient internalization of SPIO for cellular imaging. However, the potential hazards of transfection agents are not fully investigated. Instead of SPIO, we used commercially available new tagging material, fluorescent magnetic nanoparticle (MNP) containing rhodamine B isothiocyanate within a silica shell (Biterials, Seoul, Korea). This tagging material does not require transfection agents for the cell labeling. In addition to that, the core of this MNP is composed of ferrite and the inner portion of silica shell contains fluorescent materials, therefore, it has both magnetic and optical features. This study was designed to track intrasplenically injected bone marrow mesenchymal stem cells (MSCs) labeled with fluorescent MNP in liver cirrhosis rat model with 3-T magnetic resonance equipment. We compared magnetic resonance imaging (MRI) of livers in rats which were injected with non-labeled stem cells or labeled stem cells with MNP or SPIO. We found that the respective liver-to-muscle contrast-to-noise ratios at 3 and 5 h after MNP or SPIO-labeled stem cell injection was significantly lower than that of pre-injection and non-labeled group. There was no significant difference between MNP-labeled group and SPIO-labeled group. We can effectively detect intrasplenically injected MNP-labeled MSCs in an experimental rat model of liver cirrhosis with 3-T MRI.     

量子点的荧光特性在生物探针方面的应用

量子点具有传统有机荧光染料无可比拟的光学魅力,在生物医学及材料领域已引起广泛的兴趣,许多科学工作者在量子点用于生物学领域方面已经取得一定进展。目前,量子点最有前途的应用领域是在生物体系中作为荧光标记物。通过观察量子点标记分子与靶分子相互作用的部位,及其在活细胞内的运行轨迹,可能为信号传递的分子机制提供线索,从而为阐明细胞生长发育的调控及癌变规律提供直观依据。文章介绍了量子点研究生物大分子之间的相互作用、生物大分子荧光标记、细胞及生物组织的荧光标记与成像以及活体成像等方面的应用。并概述了纳米量子点作为生物荧光探针的应用前景以及亟待解决的问题。     

Rapid and recoverable in vivo magnetic resonance imaging of the adult zebrafish at 7T

Increasing scientific interest in the zebrafish as a model organism across a range of biomedical and biological research areas raises the need for the development of in vivo imaging tools appropriate to this subject. Development of the embryonic and early stage forms of the subject can currently be assessed using optical based techniques due to the transparent nature of the species at these early stages. However this is not an option during the juvenile and adult stages when the subjects become opaque. Magnetic resonance imaging (MRI) techniques would allow for the longitudinal and non-invasive assessment of development and health in these later life stages. However, the small size of the zebrafish and its aquatic environment represent considerable challenges for the technique. We have developed a suitable flow cell system that incorporates a dedicated MRI imaging coil to solve these challenges. The system maintains and monitors a zebrafish during a scan and allows for it to be fully recovered. The imaging properties of this system compare well with those of other preclinical MRI coils used in rodent models. This enables the rapid acquisition of MRI data which are comparable in terms of quality and acquisition time. This would allow the many unique opportunities of the zebrafish as a model organism to be combined with the benefits of non-invasive MRI.     

Fast voice-coil scanning optical-resolution photoacoustic microscopy

We developed a photoacoustic imaging system that has real-time imaging capability with optical resolution. The imaging system is capable of scanning at 20 Hz over a 9 mm range and up to 40 Hz over a 1 mm scanning range. A focused laser beam provides a lateral resolution of 3.4 μm as measured in an optically nonscattering medium. Flows of micrometer-sized carbon particles or whole blood in a silicone tube and individual red blood cells (RBCs) in mouse ear capillaries were also imaged in real time, demonstrating the capability to image highly dynamic processes in vivo at a micrometer-scale resolution.     

Magnetoplex based on MnFe<Subscript>2</Subscript>O<Subscript>4</Subscript> nanocrystals for magnetic labeling and MR imaging of human mesenchymal stem cells

For efficient labeling and tracking via magnetic resonance (MR) imaging of human mesenchymal stem cells (h-MSCs), magnetic labeling agents must be responsive to an external magnetic field. Thus, we developed ultrasensitive magnetoplex as a magnetic labeling agent composed of PEGylated MnFe₂O₄ nanocrystals (PMNCs) and polycationics (poly-l-lysine, PLL) for efficient labeling of the h-MSCs and monitoring of the transplanted h-MSCs for a long term. PMNCs were prepared by nanoemulsion methods composed of MnFe₂O₄ nanocrystals (MNCs) and amphiphilic polymers (mPEG–dodecanoic acid). The prepared PMNCs exhibited excellent biocompatibility and their polycationic complexes (PMNCs/PLL) demonstrated remarkable sensitivity compared with magnetic iron oxide nanoparticles (MION)/PLL or Ferumoxides/PLL. Furthermore, PMNCs demonstrated the potentials for novel diagnostic and therapeutic strategies with potential applications in various biomedical fields.     

基于ICCD的快速荧光显微成像技术及在活细胞研究中的初步应用

利用像增强型CCD、氩离子激光器和氙灯等建立了一套快速荧光显微成像系统,并初步应用于活细胞研究。实时观测和拍摄了大鼠脑微血管内皮细胞增殖分裂过程中细胞内钙敏感荧光探针Fluo-3标记的钙离子浓度分布快速变化的图像,并选取动态图像中四个典型的点给出荧光强度灰度值的变化曲线。该系统可用于高灵敏实时记录活细胞内基于荧光显微成像的快速变化过程,为活细胞的研究提供了一种很好的观察和分析手段。     

2-NBDG Fluorescence Imaging of Hypermetabolic Circulating Tumor Cells in Mouse Xenograft model of Breast Cancer

Objectives

To determine use of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) as a tracer for detection of hypermetabolic circulating tumor cells (CTC) by fluorescence imaging.

Procedures

Human breast cancer cells were implanted in the mammary gland fat pad of athymic mice to establish orthotopic human breast cancer xenografts as a mouse model of circulating breast cancer cells. Near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DeoxyGlucosone 750 (2-DG 750) was conducted to assess glucose metabolism of xenograft tumors. Following incubation with fluorescent 2-NBDG, circulating breast cancer cells in the blood samples collected from the tumor-bearing mice were collected by magnetic separation, followed by fluorescence imaging for 2-NBDG uptake by circulating breast cancer cells, and correlation of the number of hypermetabolic circulating breast cancer cells with tumor size at the time when the blood samples were collected.

Results

Human breast cancer xenograft tumors derived from MDA-MB-231, BT474, or SKBR-3 cells were visualized on near-infrared fluorescence imaging of the tumor-bearing mice injected with 2-DG 750. Hypermetabolic circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in the blood samples from tumor-bearing mice and visualized by fluorescence imaging, but not in the blood samples from normal control mice. The number of hypermetabolic circulating breast cancer cells increased along with growth of xenograft tumors, with the number of hypermetabolic circulating breast cancer cells detected in the mice bearing MDA-MB231 xenografts larger than those in the mice bearing BT474 or SKBR-3 xenograft tumors.

Conclusions

Circulating breast cancer cells with increased uptake of fluorescent 2-NBDG were detected in mice bearing human breast cancer xenograft tumors by fluorescence imaging, suggesting clinical use of 2-NBDG as a tracer for fluorescence imaging of hypermetabolic circulating breast cancer cells.     

准连续性动脉自旋标记技术在磁共振系统上的序列实现和参数优化

准连续性动脉自旋标记技术(pCASL)是一种新兴的动脉自旋标记脑灌注成像技术(ASL)：一方面,它克服了连续性动脉自旋标记技术(CASL)需要独立发射线圈的硬件限制;另一方面,也避免了脉冲式动脉自旋标记技术(PASL)带来的标记效率低的影响．为了在 1.5 T 磁共振系统上开发一款可稳定应用于临床扫描的 pCASL 序列;并使用该序列准确获得反
应灌注功能的局部脑血流量值(Regional Cerebral Blood Flow, rCBF)．该文利用水模测试pCASL 序列,验证了标记部分的标记性能并通过人体实验,优化了协议中标记位置中心到成像层面中心的距离和标记部分结束点到成像脉冲开始前的等待时间这两项参数．基于优化了参数的 pCASL 协议,扫描 12 组正常志愿者,观测灌注信号分布情况,并对特定灰质区域定量计算,对比不同个体该区域的 rCBF 值．通过人体实验,经验性地确定了延迟时间为 1 200 ms、标记距离为 70 mm 时灌注图像的信噪比达到最优．将两项优化后的参数存入协议中,并使用协议扫描,共获取 12 组结果,其中的 10 组都表明灌注信号稳定均匀,并且灰质区域的 CBF 值同经验结果一致．该工作在1.5 T 的磁共振系统上成功实现了 pCASL序列,经优化参数后的协议扫描,可以获得准确稳定的脑部灌注信号．
     

A comparison study between the saturation-recovery-T1 and CASL MRI methods for quantitative CBF imaging

The saturation-recovery (SR)-T₁ MRI method for quantitatively imaging cerebral blood flow (CBF) change (ΔCBF) concurrently with the blood oxygenation level dependence (BOLD) alteration has been recently developed and validated by simultaneous measurement of relative CBF change using laser Doppler flowmetry (LDF) in rats at 9.4T. In this study, ΔCBF induced by mildly transient hypercapnia and measured by the SR-T₁ MRI method was rigorously compared with an established perfusion MRI method—continuous arterial spin labeling (CASL) approach in normal and preclinical middle cerebral artery occlusion (MCAo) rat models. The results show an excellent agreement between ΔCBF values measured with these two imaging methods. Moreover, the intrinsic longitudinal relaxation rate (R₁^int) was experimentally determined in vivo in normal rat brains at 9.4T by comparing two independent measures of the apparent longitudinal relaxation rate (R₁^app) and CBF measured by the CSAL approach across a wide range of perfusion. In turn, the R₁^int constant can be employed to calculate the CBF value based on the R₁^app measurement in healthy brain. This comparison study validates the fundamental relationship for linking brain tissue water R₁^app and cerebral perfusion, demonstrates the feasibility of imaging and quantifying both CBF and its change using the SR-T₁ MRI method in vivo.     

基于同步移相显微干涉的血红细胞形貌测量方法

血红细胞的形貌特征是医学领域对多种疾病进行预防和诊断的一项重要指标,提出一种同步移相显微干涉法实现对血红细胞形貌的动态测量。搭建了透射式显微干涉成像系统,测量了100 μm内径模拟微血管内、名义直径为7 μm～8 μm、高度最大值为2 μm的新西兰兔血红细胞,针对血红细胞所处的微血管环境提出了基于微血管相位相减的血红细胞形貌提取方法和成像放大率校正方法,实验得到模拟微血管内的血红细胞平均直径7.757 μm和平均最高高度2.022 μm,验证了本方法具有在体定量测量血红细胞形貌的潜力。