Polychrome labeling of bone with seven different fluorochromes: Enhancing fluorochrome discrimination by spectral image analysis
Polychrome labeling of bone with seven different fluorochromes: Enhancing fluorochrome discrimination by spectral image analysis
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DOI：
10.1016/j.bone.2005.05.008
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发表时间：
2005-10-01
期刊：
影响因子：
4.1
通讯作者：
Kolk, A
中科院分区：
文献类型：
--
作者：
Pautke, C;Vogt, S;Kolk, A
体内骨形成和重塑可以通过钙结合荧光染料的多色标记来评估。然而，荧光染料的数量限制了这种技术，因为随着数量的增加，荧光光谱不可避免地重叠，这使得鉴别更加困难。为了提高识别能力，我们对光谱图像进行了分析。以雄性Wistar大鼠股骨非临界尺寸缺损为骨形成模型。从术后第4天开始，每3天皮下注射8种不同的荧光色素（钙黄蛋白蓝、二甲酚橙、钙黄蛋白、茜素络合酮、多西环素、罗利特环素、血卟啉和BAPTA）。随后，将骨标本包埋在甲基丙烯酸甲酯中，并使用Sagnac型干涉仪（ASI，以色列）进行光谱图像采集分析。应用的8种荧光染料中有7种可以用光谱图像检查来分辨。利用BAPTA，我们提出了一种适用于骨标记的新型荧光着色剂。由于光谱图像采集的优越灵敏度，可以减少骨切片的厚度，从而可以分析5pm厚的切片。光谱分解和随后的线性分解允许在不受任何其他干扰的情况下描述每个单独的荧光色，从而能够对标记区域进行可靠和卓越的形态计量学分析。(c) 2005爱思唯尔公司版权所有。
Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel).Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrorne suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 pm thick sections Could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrorne without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions. (c) 2005 Elsevier Inc. All rights reserved.