Putman C A, De Grooth B G, Wiegant J, Raap A K, Van der Werf K O, Van Hulst N F, Greve J
Department of Applied Physics, University of Twente, Enschede, The Netherlands.
Cytometry. 1993;14(4):356-61. doi: 10.1002/cyto.990140403.
Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1-79 results in clear marking of the telomere region 1p36. The diameter of the probe p1-79 linked reaction product was 75-100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material.
原子力显微镜（AFM）能够以高空间分辨率生成被研究样品的形貌图。我们推测，那些能产生可根据其形貌特性与周围物质区分开来的反应产物的细胞化学染色技术，在AFM中是适用的。在此，我们通过采用一种原位杂交技术来证明这一推测的正确性，该技术中最终的标记物是过氧化物酶/二氨基联苯胺反应的沉淀产物。在识别人类染色体1（1q12）异染色质区域的DNA探针pUC1.77杂交后，AFM能清晰地检测到原位杂交位点。用DNA探针p1 - 79进行原位杂交可清晰标记端粒区域1p36。与探针p1 - 79相连的反应产物的直径为75 - 100纳米，这表明通过这种AFM DNA测绘方法很容易达到200纳米的分辨率。这种精度与沉淀物质的量直接相关。
