微血管中场流的空间时间分析
Summary
为了量化高速毛细管流图像序列中的微血管流，我们开发了STAFF（场流空间时间分析）软件。在整个图像字段和时间上，STAFF 评估流速并生成一系列颜色编码的空间地图，用于可视化和表格输出，以便进行定量分析。
Abstract
血流速度和分布的变化对于维持组织和器官灌注以响应不同的细胞需求至关重要。此外，微循环缺陷的出现可能是多种疾病发展的主要指标。光学成像技术的进步使生命内显微镜（IVM）成为一种实用的方法，允许在细胞和亚细胞水平上对活的动物进行高速成像。然而，尽管保持足够的组织灌注的重要性，毛细管流动的空间和时间变异性很少被记录。在标准方法中，在有限的时间内选择少量毛细管段进行成像。为了以无偏的方式全面量化毛细管流，我们开发了基于场流的空间时态分析 （STAFF），这是 FIJI 开源图像分析软件的宏。STAFF 使用毛细血管内满场血流的高速图像序列，生成表示每个血管段每个时间间隔的称为 kymograph 的随时间运动的图像。STAFF 从 kymograph 计算红血球随时间移动的距离的速度，并将速度数据输出为一系列颜色编码的空间地图，用于可视化和表格输出，用于定量分析。在正常小鼠肝脏中，STAFF 分析定量了球状内近中心区域和近层区域之间的流动速度的深刻变化。更出乎意料的是，并排的正弦和数秒内在单个血管段内出现的波动之间，在流速上出现的差异。STAFF 是一种功能强大的新工具，能够通过测量毛细管流的复杂时空动力学来提供新颖的见解。
Introduction
微血管在生理学中起着至关重要的作用，确保在不断变化的条件下组织的有效灌注。微血管功能障碍与多种疾病有关，包括长期心血管发病率和死亡率、痴呆症的发展以及肝肾疾病，因此是生物医学调查1、2、3、4、5中关注的关键因素。虽然已使用多种技术来评估组织灌注，但只有生命内显微镜能够以必要的时间和空间分辨率收集数据，以表征单个毛细血管级别的血流。
微血管流可以通过荧光微球的运动或在膜内荧光标记（例如荧光标记的dextran或白蛋白）6、7的背景下红血球的运动在荧光显微镜中可视化。微血管流可以使用宽场显微镜在表面细胞层中成像，也可以使用共聚焦或多光子显微镜在深度成像。然而，毛细管流速使红血球的通过一般不能以低于60帧/秒的速度捕获。由于大多数激光扫描共聚焦和多光子显微镜需要1~5s扫描完整图像场，这种速度通常只能通过限制视场来实现，有时只能限制一个扫描线8。将测量限制在选定的毛细管段 （1） 的过程可能会引入选择偏差，（2） 使得无法捕获毛细管血流速率的空间和时间异质性。相比之下，毛细管网络的图像可以收集超过100fps的速度使用广域数字显微镜配备科学互补金属氧化物半导体（sCMOS）相机9，10。这些廉价的系统，在典型的生物医学实验室中很常见，使得在整个二维网络中成像微血管流成为可能，基本上都是连续的。问题就变成了找到一种分析方法，该方法能够从高速视频显微镜生成的大量复杂图像数据中提取有意义的定量数据。
为了能够分析全场流数据，我们开发了STAFF，新的图像分析软件，可以连续测量整个显微镜领域的微血管流量，以高速收集图像系列11。该方法与各种不同的实验系统和成像模式兼容，STAFF图像分析软件作为FIJI实现ImageJ12的宏观工具集而实现。这里用来可视化微血管流动的基本原理是，首先，必须提供一些对比度才能成像毛细血管内的红血球。在我们的研究中，对比是由红血球排除的散装荧光探针提供的。然后，流速可以从红血球的位移中量化，红血球在荧光标记的血浆中显示为负斑，图像从活的动物8高速收集。然后，我们使用STAFF在称为kymograph的多个时间间隔内沿每个毛细管段绘制距离图，然后检测Kymograph13中存在的斜坡，并从这些斜坡上计算微血管流速。该方法可应用于从任何毛细管床上采集的图像，这些图像可用于成像。在这里，我们描述了IVM和STAFF在肝脏血流研究中的应用。
Protocol
Representative Results
Discussion
此协议中有多个关键步骤。首先，在肝脏的术中成像过程中尽量减少运动对于生成可用于使用 STAFF进行毛细管流量分析的短片至关重要。由于隔膜的接近，呼吸引起的运动时间很短，每次呼吸后，固定肝脏将恢复到初始位置。使用纱布将手术暴露的肝脏固定在盖板底盘上，然后用倒置显微镜从下面成像，用于在呼吸16、17、18、19之间固定器官。<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这里提出的研究得到了国家卫生研究院（NIH U01 GM111243和NIH NIDDK P30 DK079312）的资助。在印第安纳州生物显微镜中心进行了生命内显微镜研究。我们感谢马尔戈扎塔·卡莫卡博士在显微镜方面提供技术支持。
Materials
| #5 forceps | Fine Science Tools | 11251-20 | Dumont #5 Inox Forceps |
| C57BL/6 mice | Jackson Labs | male 9-12 weeks old | |
| Cannula | Instech | BTPE-10 | Polyethylene Tubing .011x.024in |
| CMOS camera | Hamamatsu | C11440-42U30 | 4.0LT Scientific CMOS |
| Coverslip-bottomed dish | Electron Microscopy Sciences | WillCo Dish glass bottom GWST5040 | |
| Dissecting scissors | Fine Science Tools | ||
| Fiji ImageJ Image analysis software | https://fiji.sc/ ; https://downloads.imagej.net/fiji/Life-Line/fiji-win64-20170530.zip | ||
| Fluorescein dextran | Thermo Fisher, Invitrogen | D1822 | Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable |
| Gauze sponge | Fisher | 22-415-504 | 2×2 inch Dukal sterile gauze sponges |
| Heating pad | Reptitherm | RH-4 | between mouse and stage |
| Heating pad | Sunbeam | 000732-500-000U | over mouse |
| Inverted epifluorescence microscope | Nikon | Nikon TiE inverted microscope | |
| Isis Rodent electric shaver | Braun Aesculap | GT420 | |
| Isofluorane | Abbott GmbH | PZN4831850 | |
| Luer stub adapter | Fisher | 14-826-19E | Catheter adapter |
| Micro scissors | Castro Viejo | ||
| Microscope objective | Nikon | Plan Fluor 20x, NA 0.75 water immersion | |
| Needle | Fisher | 30 Ga.x1/2" | |
| Needle holder | Olsen-Hegar | ||
| Objective heater | BioScience Tools | MTC-HLS-025 | Temperature controller with objective heater |
| Rectal thermometer | Braintree Scientific, INC | TH-5A | Mouse Body Temperature monitoring |
| STAFF macros | https://github.com/icbm-iupui/STAFF | ||
| Suture string | Harvard Bioscience | 723288 | silk black suture, 6-0, spool |
Riferimenti
- Shen, J., et al. Subclinical Vascular Dysfunction Associated with Metabolic Syndrome in African Americans and Whites. The Journal of clinical Endocrinology and Metabolism. 100 (11), 4231-4239 (2015).
- Sherman, I. A., Pappas, S. C., Fisher, M. M. Hepatic microvascular changes associated with development of liver fibrosis and cirrhosis. American Journal of Physiology-Heart and Circulatory Physiology. 258 (2), 460-465 (1990).
- Zafrani, L., Ince, C. Microcirculation in Acute and Chronic Kidney Diseases. American Journal of Kidney Diseases. 66 (6), 1083-1094 (2015).
- Nielsen, R. B., et al. Capillary dysfunction is associated with symptom severity and neurodegeneration in Alzheimer’s disease. Alzheimer’s & Dementia. 13 (10), 1143-1153 (2017).
- Houben, A. J. H. M., Martens, R. J. H., Stehouwer, C. D. A. Assessing Microvascular Function in Humans from a Chronic Disease Perspective. Journal of the American Society of Nephrology. 28 (12), 3461-3472 (2017).
- Clemens, M., Zhang, J. Regulation of Sinusoidal Perfusion: In Vivo Methodology and Control by Endothelins. Seminars in Liver Disease. 19 (04), 383-396 (1999).
- Uhlmann, S., Uhlmann, D., Spiegel, H. U. Evaluation of hepatic microcirculation by in vivo microscopy. Journal of investigative surgery : the official journal of the Academy of Surgical Research. 12 (4), 179-193 (1999).
- Dunn, K. W., Sutton, T. A., Sandoval, R. M. Live-animal imaging of renal function by multiphoton microscopy. Current protocols in cytometry. , (2012).
- von Diezmann, A., Shechtman, Y., Moerner, W. E. Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking. Chemical Reviews. 117 (11), 7244-7275 (2017).
- Huang, Z. -. L., et al. Localization-based super-resolution microscopy with an sCMOS camera. Optics Express. 19 (20), 19156 (2011).
- Clendenon, S. G., et al. A simple automated method for continuous fieldwise measurement of microvascular hemodynamics. Microvascular Research. 123, 7-13 (2019).
- Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9 (7), 676-682 (2012).
- Liu, Z. -. Q. Scale space approach to directional analysis of images. Applied Optics. 30 (11), 1369 (1991).
- Linkert, M., et al. Metadata matters: access to image data in the real world. The Journal of cell biology. 189 (5), 777-782 (2010).
- Sherman, I. A., Pappas, S. C., Fisher, M. M. Hepatic microvascular changes associated with development of liver fibrosis and cirrhosis. American Journal of Physiology-Heart and Circulatory Physiology. 258 (2), 460-465 (1990).
- Babbey, C. M., et al. Quantitative intravital microscopy of hepatic transport. IntraVital. 1 (1), 44-53 (2012).
- Dunn, K. W., Ryan, J. C. Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats. Methods. 128, 40-51 (2017).
- Ryan, J., et al. Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition. Drug metabolism and disposition: the biological fate of chemicals. 46 (5), 704-718 (2018).
- Sandoval, R. M., Molitoris, B. A. Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats. Journal of Visualized Experiments. (74), e50052 (2013).
- Chhatbar, P. Y., Kara, P. Improved blood velocity measurements with a hybrid image filtering and iterative Radon transform algorithm. Frontiers in Neuroscience. 7, 106 (2013).
- Drew, P. J., Blinder, P., Cauwenberghs, G., Shih, A. Y., Kleinfeld, D. Rapid determination of particle velocity from space-time images using the Radon transform. Journal of computational neuroscience. 29 (1-2), 5-11 (2010).
- Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proceedings of the National Academy of Sciences of the United States of America. 95 (26), 15741-15746 (1998).
- Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. -. L. Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics. Journal of visualized experiments : JoVE. (101), e52303 (2015).
- Hoshikawa, R., et al. Dynamic Flow Velocity Mapping from Fluorescent Dye Transit Times in the Brain Surface Microcirculation of Anesthetized Rats and Mice. Microcirculation. 23 (6), 416-425 (2016).
- Sironi, L., et al. In vivo flow mapping in complex vessel networks by single image correlation. Scientific reports. 4 (1), 7341 (2014).
- Kamoun, W. S., et al. Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nature Methods. 7 (8), 655-660 (2010).
