Timmel Tobias, Schuelke Markus, Spuler Simone
1 Muscle Research Unit, Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine Berlin, Lindenberger Weg 80, D-13125 Berlin, Germany.
2 Department of Neuropediatrics and NeuroCure Clinical Research Center, Charité Universitätsmedizin, Augustenburger Platz 1, D-13353 Berlin, Germany.
Microsc Microanal. 2014 Apr;20(2):514-20. doi: 10.1017/S1431927613014098. Epub 2014 Feb 13.
Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.
将荧光显微镜的生物特异性与原子力显微镜（AFM）揭示的形貌特征相结合，为细胞生物学提供了新的见解。然而，缺乏系统的对齐能力，特别是在扫描探针AFM中，由于AFM漂移导致图像失配随时间增加，限制了这种联合应用方法。我们提出了一种使用悬臂尖端作为参考标志的对齐校正方法。由于在荧光图像和AFM图像中都知道精确的尖端位置，因此精确重新对齐成为可能。我们使用珠子在复杂的人工样品中证明了该方法的有效性。然后，我们将此方法扩展到生物样品，以描绘固定和活的人类成纤维细胞中的膜结构。我们能够将纳米级膜结构（如网格蛋白包被小窝）映射到它们各自的荧光点。荧光信号与地形结构之间的可靠对齐为评估细胞表面的关键生物过程（如内吞作用和胞吐作用）开辟了可能性。
