Allen M J, Bradbury E M, Balhorn R
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616, USA.
J Struct Biol. 1995 May-Jun;114(3):197-208. doi: 10.1006/jsbi.1995.1019.
We used atomic force microscopy (AFM), which utilizes a novel 3D image-contrast mechanism, to obtain nanometer-resolved, topographic data images of the natural surface structures of untreated bovine sperm cells. Freshly ejaculated, thawed, sonicated, and demembranated bovine sperm were adsorbed passively or by motility from suspension onto a coverglass substrate and directly imaged in normal air and saline environments without damaging the cells. Our AFM images of the surface structures of unfixed sperm imaged in normal air were consistent with previous electron microscope results on frozen or fixed sperm, demonstrating that the accurate preservation of small cellular structures is achievable using greatly simplified AFM sample preparation and imaging environments. Our AFM results also indicate that imaging sperm in physiologic buffer provides more native views of sperm due to the retention of cytoplasmic structures easily disrupted by drying forces. In addition, the AFM images show that numerous nanometersized subcellular structures of the sperm head and tail regions could be clearly visualized on rapidly prepared, unfixed, intact cells. Consequently, AFM should be considered a new tool for studying sperm structure abnormalities and monitoring the specific effects of, or damage caused by, various chemical reactants or other treatments on the structures of metabolically active or partially demembranated sperm. AFM is now emerging as an important new structural technique for imaging hydrated cells and organelles and, in addition, has the capabilities to physically "interrogate" them with the local probe.
我们使用了原子力显微镜（AFM），它利用一种新颖的三维图像对比机制，来获取未处理的牛精子细胞自然表面结构的纳米级分辨率地形数据图像。将新鲜射出、解冻、超声处理和去膜的牛精子通过被动吸附或利用其运动性从悬浮液中吸附到盖玻片基质上，并在正常空气和盐水环境中直接成像，而不会损伤细胞。我们在正常空气中对未固定精子的表面结构进行的AFM图像与先前关于冷冻或固定精子的电子显微镜结果一致，这表明使用大大简化的AFM样品制备和成像环境能够准确保存微小的细胞结构。我们的AFM结果还表明，在生理缓冲液中对精子进行成像能提供更接近精子天然状态的视图，因为干燥力容易破坏的细胞质结构得以保留。此外，AFM图像显示，在快速制备的、未固定的完整细胞上，可以清晰地看到精子头部和尾部区域众多纳米级的亚细胞结构。因此，AFM应被视为研究精子结构异常以及监测各种化学反应物或其他处理对代谢活跃或部分去膜精子结构的特定影响或造成的损伤的一种新工具。AFM目前正成为一种用于对水合细胞和细胞器进行成像的重要新结构技术，此外，它还具备用局部探针进行物理“探测”的能力。
