This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

这项研究是按照新加坡的机构动物护理和使用委员会（批准的协议没有。R15-0225）的国立大学。 1.手术动物模型的制备<ol><li> 血管插管 <ol><li>麻醉雄性Sprague-Dawley大鼠（6 - 7周龄）称重（203±20）G用氯胺酮（37.5毫克/毫升）和甲苯噻嗪（5毫克/毫升）的鸡尾酒通过腹膜内（IP）注射（2毫升/千克） 。不要回顾一下针头或注射后注射器将其删除。 </li><li>一旦动物被麻醉（趾捏证实），把它放在一个加热垫，以保持其体温在37℃。轻轻地刮在肩胛骨，前宫颈，小腹，内侧后腿和阴囊的头发。轻轻地克制使用胶粘纸带的腿。 </li><li>采用显微剪刀，一边欣赏角度的，通过执行镊子所有外科手术立体显微镜。放置在一个抗穿刺托盘中的所有锋利的外科手术工具，以防止手术过程中受伤。 </li><li>在进行切开前擦洗手术部位交替碘和70％乙醇3次。同花顺30 IU / ml肝素盐水解决方案的所有导管。 </li><li>做一个1 - 使用一双手术剪过右颈静脉肩胛骨1.5厘米中线皮肤切口。钝性分离分离筋膜，露出颈静脉，用聚乙烯管（PE-50）充满利用5-0丝线缝合肝素盐水导管插入它。注入补充麻醉必要时（1/3到初始剂量的1/2，静脉内（IV））在整个手术和实验过程。 </li><li>执行气管切开，以保持呼吸道通畅。做一个1 - 在颈前区有1.5厘米的切口。使用的聚乙烯管（PE-205）用2-0丝线缝合以固定导管代替导管插入气管。 </li><li>监测血压通过在股动脉插管。使1 - 在后腿的左内侧表面1.5厘米切口。分离钝性分离股动脉。导管插入填充用5-0丝线缝合肝素盐水的聚乙烯管（PE-10）的股动脉。 </li></ol></li><li> 提睾肌准备和流量可视化 <ol><li>将5-0丝线穿过阴囊的顶点来扩展它。使沿阴囊腹面切口。定期应用暖等渗溶液（37℃; pH 7.4）中到暴露的肌肉。 </li><li>删除认真彻底周围结缔组织使用棉签。 </li><li>通过提睾肌的顶点将5-0丝线缝合。切缝合线成相等长度的两件，并在每一侧打结。切两个节点之间的肌肉，并轻轻拉动缝线拉伸它到一个定制的透明有机玻璃平台。固定缝合年底到具有粘性的蓝色平台。 注:彻底清除周围结缔组织的是获得最佳图像对比度的关键。 </li><li>重复步骤1.2.3至5〜6注视制成。小心取出使用高温烧灼附睾提睾肌。 Superfuse温暖等渗溶液到暴露肌肉，以防止组织的脱水。 <ol><li>围绕提睾肌，用纱布折叠块。盖上聚乙烯薄膜的露出肌肉。纱布片与薄膜形成浅盆保持为浸水显微镜物镜（ 图1A）温暖等渗溶液。 </li></ol></li><li>转移动物到一个活体显微镜（ 图1C）的动物阶段。动脉插管连接至连续压力监测（ 图1E）的一种生理数据采集系统。 </li><li>保持肌肉TEMPERATURE在35℃下与附着在动物的平台（ 图1B）下方的加热元件。放置一个温度探头肌肉旁，以提供给加热元件（ 图1D）的功率控制器负反馈。 </li><li>离开舞台上的动物15分钟以与环境平衡。 </li><li>可视化的活体显微镜下血流40倍水浸泡的目的和长时间工作冷凝器。 </li><li>选择无支链的小动脉（&lt;60微米）的基础上的RBC芯之间的清晰图像的焦点和对比度，CFL和血管壁，以便聚焦显微镜上的血管的直径平面。旋转安装到显微镜垂直对齐血管壁的相机。 </li><li>用的高速视频摄像机以3000 /秒的帧速率1秒记录血流量。保存所记录的视频作为未压缩的8位灰度AVI格式保存的图像质量。 注意:<html>推荐3000帧/秒的最小记录帧速率，以确保所述CFL测量可生理动脉流动条件下每红细胞至少执行一次。 </li><li>使用具有峰值发送一个蓝色滤色器在310 394纳米和光谱带通波长 - 510毫微米，以提高红细胞和血浆之间的对比度。 注:确保光谱通过从微观光源（100瓦卤素灯）蓝色的过滤器是低光照强度，以防止任何潜在的组织损伤。 </li><li>在实验结束时，安乐死动物用戊巴比妥钠的过量。 </li></ol></li></ol> 2.图像分析 <ol><li> 预处理的CFL宽度测量 <ol><li>打开MATLAB并运行"CFL_pre.m&#39;文件。 （这和其他MATLAB文件可以在找到IP"&gt;补充MATLAB归档）。 </li><li>点击"打开文件"，选择视频文件进行分析。 </li><li>调整"旋转"滑块垂直对齐血管壁。 注:用户可以通过选择"网格"单选按钮，显示该容器对准辅助网格线，并通过滑动"缩放"滑块来调整图像的缩放级别。 </li><li>点击"确认编辑"按钮，确认船舶对齐。 </li><li>点击"设置ROI作物"按钮来定义感兴趣区域（ROI）的区域。对准的图像将显示在一个弹出窗口。调整图像上的矩形目标，并双击以确认投资回报率。跳过此步骤如果不需要图像的裁切。 注意:仅包括在以分析从容器中的CFL宽度将ROI的单一容器中。点击"重置图片"按钮将图像恢复到其原来的形式，如果有必要。 </li><li>点击9，提取图像"按钮，提取所有编辑的视频帧到连续的位图图像（8位灰度"BMP"格式）。所提取的图像可以具有相同的名称作为所选择的视频文件的文件夹中找到。 </li></ol></li><li> CFL宽度的测量 <ol><li>打开MATLAB并运行"CFL_measure.m&#39;文件。 </li><li>点击"选择文件夹"，选择包含提取图像的文件夹。 </li><li>单击包含图片的文件夹，然后单击"选择文件夹"。该文件夹中的第一个图像帧将会被加载，并在"灰度图像"面板中显示，随着"图像直方图"面板的灰色强度直方图一起。 </li><li>从列表框中选择所需的图像帧进行分析，否则第一图像帧将被选中。 </li><li>点击"查找容器壁"，以识别图像，其在该位置确定在内部容器壁，其中从暗光强度分布的峰过渡到点亮了两个像素。 </li><li>检查&#39;中值滤波器"以中值滤波应用到图像，以减少"椒盐"噪音。 </li><li>检查&#39;自动对比度&#39;以数字方式调整图像强度，以提高图像对比度。 </li><li>选择在列表框中自动确定阈值（τ）一个阈值化的算法，将所述灰度级分为两类 - 白像素与上述τ灰度级（CFL），并用下面τ灰度级（RBC芯）黑色像素。 注:另一种方法是，使用手动阈值，如果没有自动阈值处理算法提供了一个合适的图像阈值。点击"手动"单选按钮，调节滑块定义手动阈值。 </li><li>为了测量宽度CFL的空间变化，在"像素分辨率"框中输入像素分辨率（分辨率这个实验装置为0.42微米/像素）。 </li><li>点击"计算"按钮来获得CFL宽度的空间变化。点击"导出的.csv&#39;的CFL宽度导出数据以表格的格式。 </li><li>为了测量宽度CFL在沿着容器具体分析线的时空变化，点击"时空变化"单选按钮，然后输入帧速率信息（在本实验装置使用的帧速率为3000帧/秒）。 </li><li>在&#39;开始帧&#39;和&#39;末帧&#39;盒，输入分别在第一帧和用于分析图像的最后一帧。 </li><li>选择沿滑动"分析线"滑杆容器内的分析线的位置。确认分析线，这是双方"灰度图像"和"二值图像"上所示的位置。 </li><li>点击"计算"，以获得CFL宽度的时间变化。 CLICK"导出的.csv&#39;的CFL宽度导出数据以表格的格式。 </li></ol></li></ol>

<ol>
	<li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+variations+of+cell-free+layer+width+in+arterioles.">Temporal and spatial variations of cell-free layer width in arterioles.</a> Am J Physiol Heart Circ Physiol. 293 (3), H1526-H1535 (2007).</li><li>Ong, P. K., Namgung, B., Johnson, P. C., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+erythrocyte+aggregation+and+flow+rate+on+cell-free+layer+formation+in+arterioles.">Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles.</a> Am J Physiol Heart Circ Physiol. 298 (6), H1870-H1878 (2010).</li><li>Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+uneven+red+cell+influx+on+formation+of+cell-free+layer+in+small+venules.">Effect of uneven red cell influx on formation of cell-free layer in small venules.</a> Microvasc Res. 92, 19-24 (2014).</li><li>Goldsmith, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microcirculatory+Society.">The Microcirculatory Society.</a> Eugene M. Landis Award Lecture. The Microrheology of Human-Blood. Microvasc Res. 31 (2), 121-142 (1986).</li><li>Buerk, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+We+Model+Nitric+Oxide+Biotransport?+A+Survey+of+Mathematical+Models+for+a+Simple+Diatomic+Molecule+with+Surprisingly+Complex+Biological+Activities.">Can We Model Nitric Oxide Biotransport? A Survey of Mathematical Models for a Simple Diatomic Molecule with Surprisingly Complex Biological Activities.</a> Annu Rev Biomed Eng. 3 (1), 109-143 (2001).</li><li>Tateishi, N., Suzuki, Y., Soutani, M., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dynamics+of+erythrocytes+in+microvessels+of+isolated+rabbit+mesentery:+cell-free+layer+and+flow+resistance.">Flow dynamics of erythrocytes in microvessels of isolated rabbit mesentery: cell-free layer and flow resistance.</a> J Biomech. 27 (9), 1119-1125 (1994).</li><li>Ong, P. K., Cho, S., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cell-free+layer+formation+on+NO/O2+bioavailability+in+small+arterioles.">Effects of cell-free layer formation on NO/O2 bioavailability in small arterioles.</a> Microvasc Res. 83 (2), 168-177 (2012).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+NO+bioavailability+by+temporal+variation+of+the+cell-free+layer+width+in+small+arterioles.">Modulation of NO bioavailability by temporal variation of the cell-free layer width in small arterioles.</a> Ann Biomed Eng. 39 (3), 1012-1023 (2011).</li><li>Park, S. W., Intaglietta, M., Tartakovsky, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+stochastic+fluctuations+in+the+cell+free+layer+on+nitric+oxide+bioavailability.">Impact of stochastic fluctuations in the cell free layer on nitric oxide bioavailability.</a> Front Comput Neurosci. 9, 131 (2015).</li><li>Ng, Y. C., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+transient+model+for+prediction+of+arteriolar+NO/O2+modulation+by+spatiotemporal+variations+in+cell-free+layer+width.">Two-dimensional transient model for prediction of arteriolar NO/O2 modulation by spatiotemporal variations in cell-free layer width.</a> Microvasc Res. 97, 88-97 (2015).</li><li>Sriram, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+small+changes+in+hematocrit+on+nitric+oxide+transport+in+arterioles.">The effect of small changes in hematocrit on nitric oxide transport in arterioles.</a> Antioxid Redox Sign. 14 (2), 175-185 (2011).</li><li>Hightower, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+cardiovascular+regulation+by+the+blood/endothelium+cell-free+layer.">Integration of cardiovascular regulation by the blood/endothelium cell-free layer.</a> Wiley Interdiscip Rev Syst Biol Med. 3 (4), 458-470 (2011).</li><li>Ng, Y. C., Namgung, B., Leo, H. L., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+aggregation+may+promote+uneven+spatial+distribution+of+NO/O+in+the+downstream+vessel+of+arteriolar+bifurcations.">Erythrocyte aggregation may promote uneven spatial distribution of NO/O in the downstream vessel of arteriolar bifurcations.</a> J Biomech. , (2015).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+the+cell-free+layer+width+may+enhance+NO+bioavailability+in+small+arterioles:+Effects+of+erythrocyte+aggregation.">Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles: Effects of erythrocyte aggregation.</a> Microvasc Res. 81 (3), 303-312 (2011).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+rheology+in+microcirculation.">Erythrocyte rheology in microcirculation.</a> Jpn J Physiol. 46 (1), 1-14 (1996).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels+-+Influence+of+Erythrocyte+Aggregation.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels - Influence of Erythrocyte Aggregation.</a> Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-based+method+for+determination+of+the+cell-free+layer+width+in+microcirculation.">A computer-based method for determination of the cell-free layer width in microcirculation.</a> Microcirculation. 13 (3), 199-207 (2006).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels.</a> Influence of Erythrocyte Aggregation. Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Namgung, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+histogram-based+thresholding+methods+for+the+determination+of+cell-free+layer+width+in+small+blood+vessels.">A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels.</a> Physiol Meas. 31 (9), N61-N70 (2010).</li><li>Ong, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+method+for+cell-free+layer+width+determination+in+small+arterioles.">An automated method for cell-free layer width determination in small arterioles.</a> Physiol Meas. 32 (3), N1-N12 (2011).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Syst., Man, Cybern. 9 (1), 62-66 (1979).</li><li>Prewitt, J. M., Mendelsohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+cell+images.">The analysis of cell images.</a> Ann N Y Acad Sci. 128 (3), 1035-1053 (1966).</li><li>Ridler, T. W., Calvard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+Thresholding+Using+an+Iterative+Selection+Method.">Picture Thresholding Using an Iterative Selection Method.</a> IEEE Trans. Syst., Man, Cybern. 8 (8), 630-632 (1978).</li><li>Shanbhag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Information+Measure+as+a+Means+of+Image+Thresholding.">Utilization of Information Measure as a Means of Image Thresholding.</a> Cvgip-Graph Model Im. 56 (5), 414-419 (1994).</li><li>Bishop, J. J., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+erythrocyte+aggregation+and+venous+network+geometry+on+red+blood+cell+axial+migration.">Effects of erythrocyte aggregation and venous network geometry on red blood cell axial migration.</a> Am J Physiol Heart Circ Physiol. 281 (2), H939-H950 (2001).</li><li>Yamaguchi, S., Yamakawa, T., Niimi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+plasma+layer+in+cerebral+microvessels.">Cell-free plasma layer in cerebral microvessels.</a> Biorheology. 29 (2-3), 251-260 (1992).</li></ol>

The authors declare no competing financial interests.

CFL宽度的测量是为了更好地理解微循环血流动力学是必不可少的。特别是，CFL宽度的测量已经在肠系膜6进行spinotrapezius 24和脑25微循环。 体内 CFL宽度的常规测量通过所记录的视频帧的人工检查仅限于估计。手动测量所需几个连续视频帧的平均肉眼识别红细胞核心和血管壁15,16的边界之前。在另一项研究中，异硫氰酸荧光素（FITC） -标记的RBC和罗丹明-B异硫氰酸（RITC）标记的血浆被用来确定在猫脑微血管25的平均CFL宽度。这些以前的测量方法是非常耗时的，需要为荧光标记，这限制了紧凑型荧光灯的Wi的空间和时间分辨率的附加步骤嗞嗞的测量。与此相反，由高速摄像机的记录耦合到有效图像分割和分析，这里展示的技术中允许与一个命令的空间分辨率（0.42微米）的紧凑型荧光灯的时空变化的定量小于一个红细胞的大小1/3000秒的时间分辨率。 提睾肌的正确的手术的准备是在确定的CFL宽度测量的精度是至关重要的。特别是，彻底去除相邻结缔组织是必不可少的，以确保在提睾肌微动脉的一个良好的聚焦。此外，测量的时间和空间分辨率取决于显微镜和照相机的规格。而较高放大倍数的物镜可提高空间分辨率，其降低的视场，而这又限制了获得的容器长度为量化CFL宽度的空间变化。因此，microscopiÇ配置可以根据本技术的具体应用进行修改。 图像分割为紧凑型荧光灯宽度测量的精确度的另一个重要因素。之间开发了各种技术，基于灰度直方图图像阈值提供了用于图像分割和分析了一个简单而有效的方法。因此，前景对象是从根据其灰度级的差的背景萃取。在理想的情况下，图像的直方图将双峰和在山谷底部的阈值是微不足道的。然而， 在体内实验图像并不总是表现出这种灰度级的配置文件。我们的结果表明图像质量和对比度如何可以影响图像的分割处理。使用的光学蓝色滤光片的显著增强了RBC和在动脉的血浆之间的对比度（ 图3），以及似乎applyin时是必不可少的克的CFL宽度测量不管算法（ 图4）基于直方图的阈值处理。这导致了明显的双峰图象直方图，它允许一个有效识别的阈值。然而，应该指出的是，即使是与来自体内图像获得双峰直方图的两个峰（局部极大值）非常不等方差和直方图的宽的山谷（局部最小值）仍可影响阈值选择（ 表1 ）。因此，适当的阈值算法的选择需要研究的基础上的图像质量和用户必须考虑每个阈值算法的局限性对在量化CFL宽度最好适用性。 作为所述CFL宽度很大程度上取决于流动条件，在整个实验过程中持续动脉压测量是至关重要的。为了确定局部流动条件下，血流的伪剪速率可以通过测量血液中的容器5的平均流速进行计算。 总之，对于外科手术制备大鼠提睾肌和这里所描述的定量图像分析的协议已被用来获取关于在体内 CFL宽度的动态变化的定量信息。在确保CFL宽度测量的精确度的主要挑战包括肌肉和图像分割，这两者已在上面讨论的正常手术准备。这种技术可容易地适用于其它微循环研究，以调查血液流变学和在各种生理和病理条件血流动力学像差。因此，这些研究结果有助于微血管的治疗方法和临床干预的未来发展。

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Intravital microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:253px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">High speed camera</td>
			<td style="width:167px;">Photron</td>
			<td style="width:119px;">1024PCI</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blue filter</td>
			<td style="width:167px;">HOYA</td>
			<td style="width:119px;">B390</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pressure sensor &#38;biopac system</td>
			<td style="width:167px;">Biopac system</td>
			<td style="width:119px;">TSD104A, MP100</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Temperature controller</td>
			<td style="width:167px;">Shimaden</td>
			<td style="width:119px;">SR 1</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Plasma Lyte A</td>
			<td style="width:167px;">Baxter</td>
			<td style="width:119px;">NDC:0338-0221</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Saline 0.9%</td>
			<td style="width:167px;">Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin (5000 IU/ml)</td>
			<td style="width:167px;">LEO</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-10 polyethylene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427400</td>
			<td>.024&#34; OD X .011&#34; ID&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-50 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427411</td>
			<td>.038&#34; OD X .023&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">113-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">106-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water circulating heating pad</td>
			<td style="width:167px;">Gaymar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">Isotemp 205</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Cotton-tipped applicators</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14225</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel clip</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14120</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
		</tr>
	</tbody>
</table>

visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

所述CFL 在体内的可视化很大程度上取决于动物的手术准备。因失血过多或延长手术时间可能使动物震惊和血流像差。使用加热垫以及手术和实验期间定制平台组织温度的维护也用于保持大鼠的生理条件的关键。通过在显微镜系统中使用一个100瓦的卤素灯，即使在实验结束时没有观察到明显的组织损伤。 图2A示出了通过在大鼠提睾肌无支链动脉，在那里可以红细胞芯和内部容器壁（ 图2C）之间可以观察到所述CFL一个典型的RBC流。在实验过程中这些部件之间的良好的对比度为确保CFL宽度测量的精度是至关重要的。图像分析的初始阶段涉及检测的内血管壁。通过获取在垂直于容器沿着分析线的光强度分布，该位置是在高峰，从暗过渡到超过两个像素（ 图2B）点亮近似。 作为RBC和紧凑型荧光灯具有不同的光透射率，在灰度级的差异可被细分为两类（二值图像）。然而，在图像的直方图的两个峰之间的精确阈值的识别可以通过图像质量差和对比度（ 图3A）的限制。为了提高红细胞和CFL，蓝色过滤器可用于（ 图3B）之间的对比。这也很明显在图4中，其中，所述红细胞芯的边界可以与使用蓝色滤光片的被更精确地确定。此外，阈值算法20-23的选择也可以影响所述CFL宽度的测量（ 图4）。它在图4A中是显而易见的，不同的阈值的算法的结果中识别不同的RBC芯边界，这可能又导致错误的CFL测量。为了更好地说明在图4B所述CFL宽度测量的阈值算法的影响，使用不同的阈值的算法获得所述CFL宽度的空间配置示于图5和表1中总结。 <img alt="图1" src="/files/ftp_upload/54550/54550fig1.jpg" /> 图 1: 活体显微系统和提睾肌准备 答:通过手术外置大鼠提睾肌乙:定制平台加热元件放置提睾肌，并在35℃ 下保持它的温度:用显微CUS系统tomized动物阶段和高速摄像机微循环血流量的提睾肌D中的可视化:负反馈温度控制器和电源E:生理数据采集系统持续压力监测。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54550/54550fig2.jpg" /> 图2:图像处理的血管壁的位置和CFL宽度 A 的测定:。在动脉RBC流的典型灰度图像（血管直径= 52微米）B:沿着分析线光强分布（面板实线） C:沿容器CFL测量结果代表。该固体和虚线箭头表示RBC芯的内部容器壁和外边缘，分别（LWB＆RWB:左，右血管壁边界，LCB和RCB:左，右RBC核心边界） <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54550/54550fig3.jpg" /> 图3:。图像对比度增强与光学滤镜蓝色无（A）和蓝色滤镜（B）得到的灰度图像的图像直方图<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54550/54550fig4.jpg" /> FIGURE 4:。（:无蓝滤光片，底排（B）:用蓝色滤镜上排（A））使用RBC核心位宽使用五个不同的阈值分割算法RBC核心和血管壁图3叠加在灰度图像的边界决心（从左至右）大津的方法，最小值法，模间法，迭代选择方法（ISODATA）和模糊熵的阈值（Shanbhag）。在实线和虚线表示内血管壁和RBC核心的外边缘，分别为。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54550/54550fig5.jpg" /> 图5:CFL宽度的空间变化对应于图4b沿左CFL宽度（A）。和右（B）血管壁，分别。 （D:在血管直径的距离） <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54550/54550table1.jpg" /> 表1:阈值和CFL宽度数据在图5 * P &lt;0.001:从大津的方法显著差异。 †P &lt;0.001:左差异显著。统计分析采用双尾配对t检验进行。

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Nanyang Technological University

Research

JoVE Journal

Biology

6.3K 次观看.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

视频: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

科研

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

鼠标提睾肌肌肉制备的微循环活体成像

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...