| Objective: To search the best preparation method of the thyroid biopsy specimens for the suitable observation of the atomic force microscope (Atomic force microscopy, AFM) .To observe membrane surface ultrastructure of the normal thyroid follicular cells , adenoid tumor cells and malignant cells with AFM , and accumulate morphological information for membrane surface ultrastructures of the thyroid normal cells and tumor cells , as a result , we may supply a guide for the differential diagnosis in the benign and malignant tumors of thyroid.Methods: Applying commonly used laboratory cell extraction methods such as pressing tissue cell method , cell impression preparation and enzyme digestion centrifugal smear method to prepare the thyroid in vivo tissue samples in order to observe suitably with AFM , we compare the cell dispersion and image definition under optical microscopy and AFM (Tapping mode) using these three preparation methods of samples above and then find out the best sample preparation method . As the normal thyroid follicular cells , adenoma cells and malignant cells for the study objects , we scan these three samples with AFM and analyze them using software analyzing system and then apply SPSS 11.5 statistical analysis software to statistic their difference in average surface mean roughness, average max peak height , average maximum depth and surface area difference .Results:H.E stainning of the thyroid cell samples that prepared by pressing tissue cell method can be seen more blood , tissue fluid ,and a lot of cells overlapping with tablets . These cells impact the observation when scanning with AFM . It is difficult to show a clear and factual AFM imaging ; H.E stainning of the thyroid cell samples that prepared by cell impression preparation can be seen a clean background, and there were a lot of single sporadic cells . It is easy to find a single cell when scanning with AFM , and there were too small impurities , clear images and little interference ; H.E stainning of the thyroid cell samples that prepared by enzyme digestion centrifugal smear method can be seen thicker background and there found a lot of bare cells and a small number of cell clumps . We can see more of impurities on the cell surface when scanning with AFM and more interference . The images are not clear.Applying AFM to observe comparably membrane surface structural changes on the normal thyroid follicular cells , adenoma cells and the malignant cells ,we found that the normal thyroid cells had a circular shape with a diameter of 10 ~ 14μm in the scanning range of 20μm, while adenoma cells and malignant cells had slightly irregular shapes , slightly larger than normal cells and a rough surface. But we can not observe even tiny structures in the scope of this observation . With the gradual narrowing in the scanning , resolution is gradually increasing and the cell membrane surface ultrastructure also gradually clear . The three are visible on the surface of rugged "mountain-like" swellings , but there are differences between them . Normal thyroid cell surface swellings had more smooth, more uniform distribution, and more regulating shapes with appropriate spacing. The average size of the swellings were 27.52×36.85nm, and the average height of Z-axis were 32.37nm ; The swellings of adenoma cell surface had more rough and had gradually uneven, irregular shapes with some disorders . The cracks between swellings were "ditch-like" and inter-communicating for each other . The average size of swellings was 54.74×41.98nm . The average height of Z-axis was 51.37nm .The depth of the cracks is -59.71nm; Cancer cells had more rough surfaces, the distribution were uneven and were very irregular shapes . some swellings were "knife-like", and other swellings were "steamed bread-like" . The alinement were very confused and the cracks between the swellings were more widened than adenoma cells and seems like "ravine" . The width and height of the swellings were significantly increased . Their average size were 67.42×54.38 nm and the average height of Z-axis were 58.86nm. The depth of the cracks is -78.04nm. Using the software analysis system of AFM to measured the important parameters of the three cell membrane surface,we found that the average normal cell surface mean roughness, the Average Max peak height,the Average Maximum depth and the surface area difference were 12.505±5.307, 25.827±3.764, -17.837±2.276 and 3.794±1.030. Adenoma cell surface measurements were 45.870±3.638 , 51.838±5.941 , -58.340±4.812 and 11.305±2.996 , and the measurements of cancer cells were 57.936±3.769, 62.582±8.106, -81.649±9.808 and and 13.351±6.734. Using SPSS 11.5 statistical analysis software to analyze the measured membrane surface mean roughness , average max peak height , average maximum depth and surface area difference data , the results told us that the three had all relatively significant differences in the average surface roughness, average max peak height , average maximum depth and surface area difference .Conclusion:Cell impression preparation method is the most suitable sample preparation method for AFM to observe the thyroid tissue cells because of its clear and factual AFM imaging; Pressing tissue cell method and enzyme digestion centrifugal smear method are not suitable for AFM to scan.It is very probable to observe thyroid follicular epithelial cells with AFM.Normal thyroid follicular epithelial cells, adenoma cells and carcinoma cells are obviously different under AFM . With the deep studying of thyroid follicular epithelial cells, we can supply clinical pathology doctors with morphologyic information in nanometer about the differential diagnosis in the benign and malignant tumors of thyroid.It is possible for us to forecast wheter the cells would malignify so that we prevent the cancer hapenning. |
