Study on Physical Properties of Cells and Bio-materials with Atomic Force Microscopy

• Main Authors: Cheng, Wann-Sheng, 鄭萬順
• Other Authors: Chang Hsien-Chang
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/41537835723895857312

碩士 === 國立成功大學 === 醫學工程研究所 === 86 === Since some Scanning Probe Microscopes (SPMs) were invented, The microcosm, even the lattices of molecule, could be demonstrated authentically. Among these SPMs, Atomic Force Microscopy (AFM) is considered the towing one, its resolution could reach 1 A and its samples could be normal materials. The most advantages are the easy preparation of samples and the ability of operating in liquid environment. By using these functions, bio-materials, such as bacteria and cells, can be on- line or off-line observed in their living environment. Except morphological observation, AFM techniques have been extended to analysis of elasticity by using sharp tip and accurate feedback control system, so that the elasticity of living cells could be analyzed under biophysical circumstance. Three topics were approached in this study, described as follows: (Ⅰ) Morphologic observations of bio-materials. We got the high- resolution images of the epithelia cells (A549) that was infected with Streptococcus pyogenes group A, and discussed the possible reasons for the changes of their morphology. Alternatively, we also investigated the adhesive behavior of Streptococcus pneumoniae to the epithelia cells (Hep-2) that had been infected with respiratory syncytial viruses (RSV). The amount of bacteria adhered on the cells was about 5 times to the cells that had not been infected with RSV. Furthermore, the entry phenomenon of these bacteria into the cells was found from 3D images. Such a finding is different from the precedent view that convinced these bacteria only to adhere on the cell surface. Moreover, we on-line observed the morphological change of macrophage (p388d1) in phosphate buffer saline (PBS) after being stimulated with lipopolysaccharide (LPS). By judging the series images, we found the cells changed their morphology in 15 minutes later after adding LPS, and recovered again after 1 hour. (Ⅱ) Elastic analysis of living cell. The elasticity of the living HEp-2 in 0.9 % saline environment was calculated about 1 to 7 kPa by considering the bending of the cantilever. The central and the edge of cells were almost the same. After 0.23 % of glutaraldehyde (GA) was added to the measuring cell, the AFM images became clear owing to the cells became harder. This identical phenomenon was shown by the measurement of the cellular elasticity with AFM. We found that the elasticity changed from 5.8 kPa to 23 kPa after adding GA 20 minutes later. This is the first time to evaluate the actual value of elasticity in liquid environment after adding GA, this development may offer a novel technique to study micro- biomechanics. (Ⅲ) Thickness Measurement of ultra-thin ploymer film. AFM was used to measure the thickness of thin film with resolution in several nm level. The principle that was used to evaluate the thickness of thin film is the different ratios of the two-segment curve by the elasticity measurement. The polyphenylenediamine (PPD) film was prepared by electropolymer- ization on the tin oxide electrode based on cyclic voltammetry (CV) scanned the voltage between 0 V to 0.9 V with 100 mV/s in phosphate buffer solution (PB solution) containing 1 mM o- phenylenediamine. In the frontal 3 times of CV scanning, the thickness of PPD film were increased apparently in this solution, however, only few increment could be found from the 4th cycle. When finished the 7 times CV scanning, this thin film was calculated by ca. 90 nm.Keywords : atomic force microscopy, cellular morphology, analysis of elasticity, film thickness.