Centre for Biospectroscopy and School of Chemistry, Monash University, 3800, Victoria, Australia.
Analyst. 2010 Mar;135(3):525-30. doi: 10.1039/b919245j. Epub 2010 Jan 29.
The effects of fixation and dehydration on the distribution of heme-based molecules inside red blood cells and the structural integrity of the cells have been investigated using Raman mapping and AFM topographic imaging. A strong correlation was observed between the thickness of the cells as determined from AFM images and the intensity of the characteristic heme bands in the Raman maps, demonstrating that heme compounds are relatively evenly distributed inside dried and fixed cells in the majority of cases. The exception occurred when cells were dried in phosphate buffered saline, where more hemichrome appears close to the periphery of the cell despite the AFM image showing a plateau like topography. Using neat formaldehyde solution as a fixative is inadequate for a complete structural preservation and results in diffusion of hemoglobin into the surrounding area. However, a mixture of formaldehyde (3%) and glutaraldehyde (0.1%) in buffer was found to be sufficient to retain the structural integrity of cells with minimal autofluorescence. This protocol was also suitable for red blood cells infected with Plasmodium falciparum parasites, and preserved the characteristic knob-like structures on the infected red blood cell surface.
使用拉曼映射和 AFM 形貌成像研究了固定和脱水对血红素分子在红细胞内的分布和细胞结构完整性的影响。从 AFM 图像确定的细胞厚度与拉曼图谱中特征血红素带的强度之间存在很强的相关性，表明在大多数情况下，干燥和固定的细胞内部血红素化合物相对均匀地分布。在细胞在磷酸盐缓冲液中干燥时，情况并非如此，尽管 AFM 图像显示出类似高原的形貌，但靠近细胞边缘出现更多的正铁血红素。使用纯甲醛溶液作为固定剂不足以完全保存结构，并且会导致血红蛋白扩散到周围区域。然而，发现缓冲液中的 3%甲醛和 0.1%戊二醛的混合物足以保持细胞结构的完整性，同时最小化自发荧光。该方案也适用于感染疟原虫的红细胞，并保留了感染红细胞表面特征性的小结状结构。
