Department of Otolaryngology, University of Tampere, Tampere, Finland.
Hear Res. 2010 Oct 1;269(1-2):1-11. doi: 10.1016/j.heares.2010.07.006. Epub 2010 Jul 24.
Fluorescent tags and fluorophore-conjugated molecular probes have been extensively employed in histological studies to demonstrate nanoparticle distribution in inner ear cell populations. However, autofluorescence that exists in the rodent cochleae disturbs visualization of the fluorescent tags and fluorophore labeling. In the present work, we aimed to improve the visualization of fluorescently tagged nanoparticles and fluorophore-labeled molecular probes by treatment with CuSO(4) to quench autofluorescence in the rat inner ear. The in vivo study was performed on eight- to nine-month-old rats using confocal laser scanning microscopy, and the in vitro study was carried out with DiI-tagged poly(ethylene glycol) and poly(capro-lactone) polymersomes and different fluorescent-labeling agents using a spectrofluorometer. The nanoparticles were intratympanically administered using either an osmotic pump or transtympanic injection. Abundant autofluorescence was detected in spiral ganglion cells (SGCs), stria marginal cells, spiral ligament fibrocytes (SL) and the subcuticular cytoplasm of inner hair cells (IHCs). Sparsely distributed faint autofluorescence was also visualized in outer hair cells (OHCs). The autofluorescence was eliminated by treatment with 1 mM CuSO(4) (in 0.01 M ammonium acetate buffer) for 70-90 min, while the fluorescent tag in the nanoparticle was absolutely preserved and the labeling fluorescence signals of the molecular probes were mostly retained.
荧光标记物和荧光染料标记的分子探针已广泛应用于组织学研究中，以展示内耳细胞群体中的纳米颗粒分布。然而，啮齿动物耳蜗中存在的自发荧光会干扰荧光标记物和荧光染料标记的可视化。在本工作中，我们旨在通过使用 CuSO4 处理来淬灭大鼠内耳中的自发荧光，从而改善荧光标记纳米颗粒和荧光染料标记分子探针的可视化。体内研究在 8 至 9 月龄大鼠上进行，使用共聚焦激光扫描显微镜，体外研究则使用 DiI 标记的聚乙二醇和聚己内酯聚合物囊泡以及不同的荧光标记试剂，使用分光荧光计进行。纳米颗粒通过渗透泵或经鼓膜内注射给药。在螺旋神经节细胞（SGC）、边缘带细胞、螺旋韧带成纤维细胞（SL）和内毛细胞的亚细胞膜中检测到丰富的自发荧光。在外毛细胞（OHC）中也观察到稀疏分布的微弱自发荧光。用 1mM CuSO4（在 0.01M 醋酸铵缓冲液中）处理 70-90 分钟可消除自发荧光，而纳米颗粒中的荧光标记物则完全保留，并且分子探针的标记荧光信号大部分保留。
