Cellular Biophysical Properties And Membrane Receptors Exploited By AFM And NSOM

Nanobiotechnology,as the cross-discipline of nanotechnology and life science,is a novel technology and approach in nanoscale life science study;it can provide many insightful views into biophysical behaviors of biomolecules and cells including the intramolecular force, intermolecular interaction,and molecule-molecule and cell-cell interaction mechanism.In this work,atomic force microscopy(AFM),near-field scanning optical microscopy,laser scanning confocal microscopy and quantum dot labeling were used to investigate the biophysical properties(the parameters including morphology,membrane nanostructures,membrane pore, friction distribution,adhesion properties,specific interaction of antigen-antibody,distribution mode of membrane receptors and so on) of T lymphocytes stimulated by phorbol dibutyrate (PDB) plus ionomycin(ION) at nanoscale.In addition,the biophysical variation of erythrocytes with the time lapse was analyzed utilizing AFM.According to the experiments,we obtained many insightful results shown as the followings:1) The nanostructures of resting and activated T lymphocytes were firstly evaluated.The results indicate that cell diameter,cell volume and the mean height of membrane surface particles of CD3+ T cells increased due to PDB plus ION stimulation;as a contrast,all biophysical parameters of CD4+ and CD8+ T cells increased after stimulation.The lateral force measurements indicate the heterogeneous distribution of friction on CD4+ and CD8~+ T cell membrane,revealing the chemical and biophysical heterogeneity;2) Nano-mechanical properties of resting T cells evidently differ from activated T cells.The results obtained in air indicate that the surface adhesion force of resting CD3~+ T cells is 1025±799.84 pN,which decreased with the lapse of activation time,and that measured at 24 hours stimulation time appears to be the lowest(243.93±167.51 pN);whereas,the adhesion force of CD4~+ T cells measured at 24 hours stimulation time is the largest(827.6±271.8 pN) among three experimental groups,24,48 and 72 hours group,and force curves of CD4~+ T cells show unobvious snap-in force;as for CD8~+ T cells,the adhesion force of activated cell distributes between 300 pN and 800 pN,which is also larger than that of resting cells,100～700 pN.When the measurements were conducted in liquid,the force curves indicate that adhesion force and nonlinear interaction between bare tip and CD4~+,CD8~+ T cells are not obvious.3) As for specific interaction of antigen-antibody of CD4+ T cells measured in situ,the results exhibit that the specific interaction force of CD4 antigen-antibody is 200-1200 pN,which is larger than that of CD69 antigen-antibody(30-210 pN);as for CD8~+ T cells,however,the specific interaction force of CD8 antigen-antibody is 918±430.6 pN,which is slightly smaller than that of CD69 antigen-antibody(1097.8±675.2 pN);moreover,the specific force curves reveal the evident nonlinear interaction between tip and cell membrane;4) Investigation of distribution mode of membrane receptors.3-D reconstruction map, grayscale map and imaging results of laser scanning confocal microscopy altogether demonstrate that both CD4 receptors and activation marker CD69 receptors non-uniformly distribute on CD4~ T cell membrane,concentrating into nano-clusters and/or micro-domains.Furthermore, near-field fluorescent images,whose highest resolution is 92 nm(FWHM),also reveal the inhomogeneous distribution of CD4 and CD69 receptors on cell membrane;the statistics further indicates that more than 75%CD4 molecules on cell membrane concentrates into nano-/microstructural domains,which is supported by the analysis of area,diameter and fluorescent intensity of near-field fluorophores.As for CD8~+ T cells,the 3-D reconstruction map and grayscale map, which imply the distribution mode/pattern of membrane receptors,also illustrate that CD8 and CD69 receptors inhomogeneously distributing on cell membrane and forming nano-/microstructural domains:5) Atomic force microscopy(AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains,potentially providing useful information for forensic investigation.Here,the time-dependent,morphological changes of red blood cells(RBC) under three conditions(including controlled,room-temperature condition,uncontrolled, outdoor-environmental condition,and controlled,low-temperature condition) were observed by AFM,as well as the cellular viscoelasticity via force-vs-distance curve measurements.With time lapse,the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition.Under the controlled low-temperature condition,however,the changes in cell volume occurred mainly due to the collapse of RBCs,and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC.In the present work,the variations of biophysical properties of Tlymphocytes in the context of activation in vitro were evaluated at nanoscale for the first time,and the obtained experimental data and information offer us new insightful views for further understanding/elucidating immune response/recognition correlated biophysical behaviors of specific/non-specific T cells.On the other hand,the AFM detections on the time-,environment(temperature/humidity)- dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in medicine/forensic medicine exmination.AFM and NSOM,as two important members of scanning probe microscopy family,play crucial roles in nano-biology study due to their acquiring capabilities of nanostructures and nano-mechanical properties.As the frontier field of nanotechnology study,nanobiotechnology has aroused scientist's extensive concerns and will provide invaluable implications and insights into biophysical behaviors/processes of biomolecules and cells at nanoscale.