Munisso Maria Chiara, Yamaoka Tetsuji
Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
Biomed Res Int. 2014;2014:196837. doi: 10.1155/2014/196837. Epub 2014 May 25.
The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.
荧光染料的可用性以及用于体内成像的光学系统的进步，激发了人们对开发新方法来研究和量化标记剂生物分布的兴趣日益浓厚。然而，尽管取得了这些巨大成就，但在确定观察到的荧光是否确实对应于组织中染料的量方面，我们仍面临重大挑战。事实上，尽管远红光和近红外光可以穿透几厘米的组织，但由于光子的弹性散射，它们会在几毫米内扩散。此外，当染料标记的寡核苷酸与阳离子载体形成稳定复合物时，会观察到染料荧光强度发生很大变化。因此，测量的荧光强度会受到组织异质性和染料强度波动的影响。因此，在本研究中，开发了一种用于荧光标记寡核苷酸的定量策略，以解决这些不利影响。我们的结果证明，在用诸如硫氰酸胍等离液剂进行有效均质化和稀释后，可以实现荧光强度的完全恢复。此外，我们证明了该方法具有灵敏度高、重现性好以及组织样品易于处理的优点。
