The Application Of Atomic Force Microscopy Imaging To Lipid Bilayer, Membrane Protein And Fibroblasts

Atomic Force Microscopy (AFM), also named Scanning Force Microscopy (SFM) or Scanning Probe Microscopy (SPM) , was invented by Binnig et al. in 1986. AFM was used to investigate the surface structure characteristics of materials in the past. Recently its use has expanded into Biological Fields very quickly. Over the last 10 years AFM has become one of the most powerful Research Tools in Biology and Biological Physics, especially in Cell Biology. AFM has several advantages:Firstly, AFM can investigate biological molecules and cells in situ. Simple sample preparation is enough if needed. Secondly, a high resolution 3D image can be obtained by AFM. Thirdly AFM imaging has a less destructive effect on samples than Electronic Microscopy.The most important thing is that AFM imaging can be carried out in air, vacuum or fluid. AFM can image biological samples in their native condition and investigate cell dynamics in real time.Here we image the lipid bilayer, membrane proteins and living cells with AFM in order to indicate its power and importance in Biological Research.Part1Objective: To image the structure of the lipid bilayer at the nanometer levelwith AFM. Methods: AFM images the lipid bilayer that was formed by thevesicle fusion method. Results: The fluidity of the lipid bilayer can be investigated with AFM in fluid. The height and topography of the lipid bilayer can be calculated and observed by AFM. Conclusion: AFM is a good tool to image the lipid bilayer.Part 2Objective: To investigate the p7 membrane protein of the Hepatitis C Virus with AFM. Methods: To prepare the p7 membrane protein on mica and in the lipid bilayer, then to image the p7 protein using tapping mode Atomic Force Microscopy. Topographical information on the Hepatitis C Virus p7 protein in the DOPC/Sphingomyelin/Cholesterol lipid bilayer was obtained for study in biological conditions by AFM. Results: The ion channel structure of the p7 protein is clearly visible. The p7 protein located in the DOPC fluid phase of the DOPC/Sphingomyelin/Cholesterol lipid bilayer can assemble in the lipid bilayer. Conclusion: High resolution images of membrane proteins on mica or in the lipid bilayer can be investigated with Atomic Force Microscopy.Part3Objective: The application of Atomic Force Microscopy (AFM) to investigate Living Cells in 3D. Methods: AFM images Living Cells including the Cytoskeleton, cellular interaction etc. Fluorescence labelled cells, after drug treatment, were investigated by Confocal Laser Scanning Microscopy. Real time images of Fibroblasts were investigated by AFM during drug treatment. Results: We showed AFM images of living Fibroblasts in fluid. Contact mode AFM of Living Cells provided precise information on the shape of cellular Processes such as lamellipodia at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells. Cytochalasin B and Latrunculin B have negative effects on the cellular cytoskeleton as can be observed both by Confocal Laser Scanning Microscopy and by Atomic Force Microscopy. Conclusion: Atomic Force Microscopy can be used to investigate cells and submembrane structures in real time. Time lapse movies produced from sequential AFM images also gave a realistic view of the cellular dynamics.