Structured Illumination Combined With Atomic Force Microscopy

Cell is the smallest unit of life. The cell will express kinds of physiological state, such as reproduction and death, affected by various biological molecules within it. Therefore, in recent years researchers have focused on the cell hyperfine structure and various physical-chemical properties of cell. They carry out research mainly including far-field super resolution imaging technology and near-field scanning imaging technology.In this background, far-field super resolution imaging techniques have been developed to break the optical diffraction limit, such as stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM). SIM uses Moire effect to encode the high frequency information into the low frequency region so as to pass the imaging system with frequency low-pass filter. Then the high frequency information is recovered through demodulation, thus the frequency spectrum is extended, therefore, improving the resolution. Compared with the other super resolution methods, SIM has the advantages of simple technical scheme, low cost, fast imaging speed, etc. And atomic force microscopy (AFM) is a kind of near-field scanning imaging technology. It is based on the principle of molecular interactions on the probe tip and the sample surface. It also has high resolution in horizontal and vertical directions (1 nm level). Meanwhile AFM can scan and image the precise hyper-fine structure in cell. Therefore, AFM is of significantly importance in the nano biological sciences research.We first propose combination of SIM with atomic force microscopy. Using this imaging technology, we can obtain the information of microstructure and function in a particular area. The main research content is as follows:(1) We modify the light source of Leica DMI6000B microscopy, to build a 2D structured illumination microscopy, with Coherent 488 LP Sapphire laser as the light source, spatial light modulator as the light modulator, using two-beam interference to produce the cosine structured light. We obtain the optimal parameters, which the current experiment device can achieve, through computer simulation and experiment. We perform experiment imaging of 100 nm fluorescent beads and Actin Green labeled cytoskeleton in fixed Hela cell, and obtain higher resolution and more details than the traditional fluorescence microscopy.(2) We propose the combination of SIM with AFM imaging system. Using this imaging system, we acquire the physical properties of cell, including the position of microfilament protein and shape. We use 100 nm fluorescent beads as sample to test our system. And we obtain high resolution and physical properties of the cell through scanning and imaging of fixed Hela cell and MCF-7 cell, respectively.