The High-resolution Image And Microscopic Mechanical Properties Of Fresh-isolated Vascular Smooth Muscle Cells Studied By Atomic Force Microscopy

Aims:1) To develop a method for good immobilization of fresh-isolated arterial smooth muscle cells,which may be fit for the imaging of atomic force microscopy(AFM).2) To acquire the high-resolution imaging of flesh-isolated smooth muscle cells by using magnetic AC mode.3) To explore the microscopic mechanical properties of fresh-isolated smooth muscle cells by force volume function using contact mode AFM.Part1 Immobilization of fresh-isolated smooth muscle cells by using micro-patterned PDMS slab.An elastomeric stamp was conventionally fabricated by a standard photolithographic technique.The micro-patterned polydimethylsiloxane(PDMS) slab with micro-poles (one is of 4×4×3μm,space between two poles is 8μm) was prepared by replica molding.Fresh-isolated smooth muscle cells were obtained by the method of two-step digestion.The surface of micro-patterned PDMS slab was coated with poly-L-lysine,then the fresh-isolated arterial smooth muscle cells were immobilized inside micro-poles array by using centrifugal method.The results from ESEM imaging indicated that fresh-isolated smooth muscle cells were trapped well inside micro-pole array.Part 2 Imaging of the living smooth muscle cells by magnetic AC mode AFMIn magnetic AC mode(MAC mode) AFM,a new type of tapping mode AFM, the background resonances are absent and signal-to-noise is improved.Better signal-to-noise means that cantilevers with much lower force-constant and much smaller vibrating amplitudes can be used.This decreases damage to the sample and preserves asperities on the probe,contributing to greatly improved resolution in liquid environments.The imaging of the living smooth muscle cells by MAC mode can acquire a high resolution up to 30 nm,agreeing well with those from ESEM imaging. Stimulation of NE or mechanical stimulus led to contraction of smooth muscle cells which indicated that MAC mode AFM could make both a high resolution image and maintaining cell contraction function with little damage of cells.Part 3 Microscopic mechanics study of smooth muscle cells by using force volume function of AFMForce volume function was performed to detect the mechanical characteristics of the tapped cells.The force curves of different regions were extracted from the force mapping data.Statistical analysis strongly indicated that cell body periphery was always stiffer than its central region.Under NE of 0.02mg/ml the stiffness of both the cell body periphery and its central region increased,and the stiffness of the cell body periphery increased more obvious than that of its central region.Summary:A new method was established,in which both morphological alternations at nanometer level and contraction function of single smooth muscle cell could be observed by AFM.Micro-pole array on the PDMS surface was used to trap fresh isolated smooth muscle cells,which may lead to a good immobilization of fresh isolated arterial smooth muscle cells;MAC mode AFM will be fit well for the imaging of living cells and force volume imaging is used to detect the mechanical properties after smooth muscle cell contraction.We supposed that our studies would result in,to some extent,an understanding of certain physical pathology details of smooth muscle cells and of the application of AFM and some physical techniques to fresh isolated cells.