Atomic Force Microscopy Analysis Of Morphology And Biomechanics Of Erythrocyte Under Cardiopulmonary Bypass

Objective:Atomic force microscope (AFM) is widely applied in the field of nanobiology,including morphological imaging, ultrastructure, mechanical properties and interaction ofbiological samples. This study is to investigate the effect of open-heart surgery andcardiopulmonary bypass on morphology and biomechanics of erythrocytes with atomicforce microscopy, and providing a new information on the changes of erythrocytes undercardiopulmonary bypass and evidence on cellular physiologic and pathologic condition byquantitative and qualitative analysis on a nanoscale.Methods:The objects of the study are venous erythrocytes collected pre-operatively (PRE group)and30min into cardiopulmonary bypass (CPB group). Both groups of Noncircularerythrocytes are counted using an fluorescence microscopy. Using AFM to conduct overalland ultrastructural examination on membrane surface of both groups of erythrocytes. WithAFM image analysis software obtains the parameters of average diameter, average height,average concavity, average roughness (Ra), root-mean-squared roughness (Rrms) andother parameters of membrane surface structure in the both groups of erythrocyte.Chemical properties of erythrocyte membrane surface are measured using AFM forcecurve. Analyzing properties of erythroeyte: depth of indentation, deformation restoringforce, adhesiveness and leap line-distance before operation and after cardiopulmonarybypass30min according to the related force-distance curve spectrums.Results:1. The ratio of noncircular erythrocytes shows no significant differences in bothgroups of erythrocyte (P>0.05).2. AFM images show that a single erythrocyte exhibited the typical one-side concave, disc shape with clear contour and smooth, soft, delicate surface in PRE group; There is anerythrocyte is relatively regular and circular, but the cell surface has obvious shallow dentswith folded edges and roughness in the central concavity in CPB group.3. There are significantly differences in average diameter, average heightand andaverage concavity in CPB group as compared with PRE group (P <0.05).4. The membrane of erythrocyte in PRE group is a rugged surface caused by bulgesof different sizes. The bulges are smooth, plump, regularly shaped and orderly arranged ina dense mesh wprk and even distribution. Gap-like concaves formed by bulges are even indepth. The bulge-shaped structure still exists on membrane surface in CPB group, but themembrane surface is relatively rough, and the bulges are irregularly shaped, disordered anduneven.5. Both of Ra and Rms in4.0μm×4.0μm and2.5μm×2.5μm measuring area weresignificantly increased in CPB group as compared with PRE group (P <0.05).6. Differences between both groups on depth of indentation and deformationrestoring force has no significant change (P <0.05), while differences in adhesiveness andleap line-distance are significantly increased in CPB group as compared with PRE group(P<0.05).7. The slope ratio of force curves has no significant differences in both groups(P>0.05).Conclusion:Cardiopulmonary bypass for30minutes induced the erythrocyte surface uplift shapederangement and membrane surface is relatively rough, and the bulges are irregularlyshaped, disordered and uneven with AFM;30min under cardiopulmonary bypassdecreased the erythrocyte average height and average concavity and increased theerythrocyte average diameter and the adhesion between cells, which could change themorphology, ultrastructure and adhesive properties of venous erythrocyte of membranesurface.