Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Blood. 2010 Oct 14;116(15):e41-55. doi: 10.1182/blood-2010-02-268466. Epub 2010 Jul 20.
In many animals, blood cell production occurs in the bone marrow. Hematopoiesis is complex, requiring self-renewing and pluripotent stem cells, differentiated progenitor and precursor cells, and supportive stroma, adipose tissue, vascular structures, and extracellular matrix. Although imaging is a vital tool in hematology research, the 3-dimensional architecture of the bone marrow tissue in situ remains largely uncharacterized. The major hindrance to imaging the intact marrow is the surrounding bone structures are almost impossible to cut/image through. We have overcome these obstacles and describe a method whereby whole-mounts of bone marrow tissue were immunostained and imaged in 3 dimensions by confocal fluorescence and reflection microscopy. We have successfully mapped by multicolor immunofluorescence the localization pattern of as many as 4 cell features simultaneously over large tiled views and to depths of approximately 150 μm. Three-dimensional images can be assessed qualitatively and quantitatively to appreciate the distribution of cell types and their interrelationships, with minimal perturbations of the tissue. We demonstrate its application to normal mouse and human marrow, to murine models of marrow failure, and to patients with aplastic anemia, myeloid, and lymphoid cell malignancies. The technique should be generally adaptable for basic laboratory investigation and for clinical diagnosis of hematologic diseases.
在许多动物中，血细胞的生成发生在骨髓中。造血是一个复杂的过程，需要自我更新和多能干细胞、分化的祖细胞和前体细胞以及支持性基质、脂肪组织、血管结构和细胞外基质。尽管成像技术是血液学研究的重要工具，但原位骨髓组织的三维结构在很大程度上仍未被描述。成像完整骨髓的主要障碍是周围的骨结构几乎不可能通过切割/成像来穿透。我们已经克服了这些障碍，并描述了一种方法，通过该方法可以通过共聚焦荧光和反射显微镜对整个骨髓组织进行免疫染色并以三维方式成像。我们已经成功地通过多色免疫荧光标记来定位多达 4 种细胞特征的模式，同时可以在大面积平铺视图和大约 150μm 的深度上进行定位。可以对三维图像进行定性和定量评估，以了解细胞类型的分布及其相互关系，而对组织的干扰最小。我们证明了它在正常小鼠和人类骨髓、骨髓衰竭的小鼠模型以及再生障碍性贫血、髓系和淋巴系细胞恶性肿瘤患者中的应用。该技术应该普遍适用于基础实验室研究和血液疾病的临床诊断。
