Atomic Force Microscopy Images Analysis In The Air And Under The Liquid Conditions

ObjectiveIn this study,we used atomic force microscopy for morphology scanning to the Al₂O₃ templates,digital optical discs,and mica film respectively in the air and liquid conditions.Analysis the difference between AFM imaging in the air and liquid condition.Optimize the working conditions of AFM in the liquid phase.Put high-resolution AFM imaging into the morphological study of biological samples,and develop the dynamic measurement mode of AFM in the liquid phase.Explore the AFM tip and the substrate functional modification program in the use of AFM to study the interaction of biological molecules.Use AFM for single biological samples such as a red blood cell morphology research study,and further develop the real-time dynamic detection technology of AFM in the solution environment of living cells such biological samples.MethodsThree groups of samples(CD-R,DVD-R and Al₂O₃) were scanned by Atomic Force Microscopy in the air and liquid conditions,in order to study their surface morphology under different conditions.Imaging natural mica and aldehyde glass-chip surface morphology using AFM,then observed the difference between surface roughness.The avidin protein molecules were modified to a new dissociation of mica surfaces and aldehyde surface,then scanning by AFM to study the avidin protein morphological characteristics.The AFM tip were silanized modified using APTES,The 3' end labeling has cy3 and 5' end amino-modified oligonucleotide probe have connected to the AFM probe tip by glutaraldehyde as a connection elements.In order to verify the effect of modification we use the inverted fluorescence confocal microscope. Fresh red blood cells in mice were spread on the freshly cleaved mica,and using the tapping mode AFM scan to study the morphological characteristics of red blood cells in mice.Results1.Comparative analysis of several solid samples topography images by AFM in air and liquid phase,we found that the morphology under the liquid condition is better than the topography image in air.Liquid conditions not only may eliminate a lot of noise signal to makes the boundaries of the raised and sunken image of the site more clearly,but also greatly reduces the tip broadening effect,so that a fine structure more prominent.2.To analysis the AFM images of nanopore template in the air and liquid phase, we found that the nano-hole diameter on the same samples is slightly different.After statistical analysis we have calculated the nanopore diameter in air and liquid were 63.04nm±5.58nm and 70.36nm±7.03nm respectively,This phenomenon may be due to the tip broadening effect in the air that both the tip and sample are exposed to the atmosphere,so that the outside interference can larger increase of the broadening effects of the needle.3.Comparative analysis the AFM images of mica and aldehyde glass chip,we can see that the freshly cleaved mica surface roughness better than aldehyde glass chip.The biggest ups and downs of the freshly cleaved mica surface is 30nm and 70nm of the aldehyde glass chip.4.The avidin protein molecules on the substrate surface imaging by the AFM was similar to the oval ball.Through image analysis of these proteins we found that the diameter of which is around 200nm～350nm.5.Mouse red blood cells in the AFM scans image shown for the rules and the center of the disc-shaped depression,and its diameter is about 5～7μm. Conclusions1.AFM images in the liquid conditions more be able to highlight the fine structure of the sample,and access to high-definition images.2.AFM probe broadening effects will affect the image quality.In the experiment we should be choice appropriate needle based on the size of sample,in order to minimize the impact of scan caused by the morphology of the needle.3.Covalent modification of the sample to the substrate surface is superior to the method of electrostatic adsorption,when the use of AFM to study the biological samples.Covalent modification can be ensure the molecule number modified to the surface,that is more conducive to study in the liquid phase.4.When use AFM to detect the interaction between biological molecules,the program of silane-modified AFM tip,then use glutaraldehyde as a connecting element to covalent modification test protein molecules to the tip(or substrate surface) is feasible.5.AFM can be achieved on biological samples such as single-cell imaging.The further development of AFM in the liquid phase under the dynamic measurement mode of great significance to biological samples,as well as the interaction between molecular biology research.