Development And Application Of Cell Membrane Research Technology Based On Second-order Nonlinear Optics

Cell membrane interface involves many physiological processes,such as molecular penetration,membrane fusion,catalytic reaction,protein adsorption and so on.It is an important research direction in the field of life science.In the past decade,many experimental techniques have been used to study cell membranes,such as molecular dynamics simulation,Fourier transform infrared spectroscopy and X-ray.Nonlinear optical technology is also widely used in cell membrane research,especially the second-order nonlinearity spectroscopy has surface-interface selectivity and has advantages in characterization of cell membrane.In this paper,a series of techniques have been developed to study the interaction between substances and cell membranes and the fusion process of model cell membranes by using second-order nonlinear optical effects.The main work includes the application of second harmonic generation(SHG)spectroscopy to measure the dynamic process of interaction between substances and cell membranes;the application of second harmonic generation(SHG)spectroscopy and sum frequency generation(SFG)spectroscopy to study the mechanism of metal ion-induced membrane fusion;and the establishment of a compatible SHG and two-photon-excited fluorescence(two-photon-excited fluorescence).The two-photon microscopic imaging system for fluorescence(TPEF)imaging can be divided into the following parts:A series of experimental methods and instruments have been developed to study the characteristics of cell membranes by using second-order nonlinear optical effects,including the surface-interface specificity of second harmonic generation(SHG)to study the interaction between substances and cell membranes,and the use of SHG and sum-frequency generation(SFG)to measure the melting of cell membranes.In addition,a two-photon microscopic imaging system compatible with SHG imaging and two-photon fluorescence imaging has been built.The specific work is divided into the following parts:Firstly,the interaction between molecule and cell membrane was studied.The second harmonic light scattering(SHS)technique based on SHG was used to study the dynamic process of the interaction between malachite green(MG)molecule and bacteria,phospholipid vesicles.SHS was used to measure the effect of Ca2+ on MG molecule penetration,and some inferences were made according to the phenomena.SHS was used to measure the effect of different concentrations of metal ions on the process of MG molecule penetration.Ca2 + and Mg2 + increased the rate of MG molecule penetration,while Cu2 + would destroy the bacterial structure and make the SHS signal rapidly drop to the initial level.On the basis of bacterial and model cell membrane experiments,SHS was successfully applied to measure the dynamic process of the interaction between penetrating peptides and animal cells.Secondly,the mechanism of membrane fusion induced by metal ions was studied by non-linear spectroscopy.We successfully measured the fusion process of Ca2+ induced membranes by designing SHG spectra with different particle sizes and different emission directions.In view of the different behavior of calcium ion,magnesium ion and negative electrophospholipid membrane,the response of phospholipid molecule to different metal ions,such as trans-complexation and cis-complexation,was measured by SFG,and the mechanism of metal ion inducing cell membrane fusion was revealed.Compared with calcium ions,phospholipids bind to Mg2 + cis-form,and the surface of phospholipids has a stronger hydration layer,and the end of phospholipids has a higher degree of order,which hinders the further fusion of cell membranes.Finally,the principle prototype of TPEF microscopic imaging system is discussed from two aspects of hardware system design and software system design.Firstly,the optical path and hardware control system of the TPEF microscopic imaging system are designed and built.The core of TPEF microscopic imaging system is laser scanning control and signal acquisition.In this paper,the galvanometer is used to control the laser scanning,and the hardware control scheme with the data acquisition card as the control core is designed.At the same time,the data acquisition card collects the output signal of the photomultiplier tube(PMT)for image reconstruction.Then,the software system of the TPEF microscopic imaging system is designed.The software system is designed and developed with Lab VIEW.The hardware system can be controlled efficiently with data acquisition card.The software system mainly realizes imaging parameters setting,galvanometer scanning mode control,PMT gain level setting,data acquisition,scanning data imaging and other functions.For the collected signal,the software embedded the script code of MATLAB to process the signal.At the end of the article,the research results are summarized and analyzed,and the continuation of the work is prospected.