The Principle Of Modulation Optical Imaging And Its Application In Biology

In the biological imaging process, auto-fluorescence from sample and the random noise generated by imaging process will lead to the acquired image blur. At present, interesting protein molecules are basically observed in over-expressed state, but this kind of practice may cause some negative results which will affect the accuracy of the experiment results, such as cell toxicity and dominant-negative, this defect limits application of some low copy gene manipulation techniques. However, the expression of protein molecules is very low and the fluorescence signal is very weak, physiologically. How to detect the weak signal from high background is the question which always haunt in researchers’ head.This paper introduces a novel method of optical imaging from the following three aspects: optical modulation imaging method.The first part mainly introduces the principle of the modulation optical imaging and introduces the corresponding two kinds of demodulation methods: 1.Optical Lock-In demodulation(OLID).2.Fourier transform demodulation(FTD). This kind of method modulate emission fluorescence through switching between two kind of lasers, so after demodulation, the weak signal can be recoverd because auto-fluorescence and random noise can not be modulated. It is very important to select suitable fluorescent protein to do experiment because of the need to modulate fluorescent protein, this part also introduces character of rsEGFP2.The second part mainly introduces the construction of TIRFM, including the multi-laser coupling, the construction of multi-functional illuminator and control the whole microscopy system using micromanager software. Because TIRFM illuminate sample by evanescent waves, illumination depth is about 100 nm, so this kind of illumination way can greatly inhibit scattering out-focus light combine with EMCCD which has the ability of detecting single photon, the whole system is very sensitive in weak signal detection.In the last part, the paper mainly introduces acquire image of actin in Hela cell and ER in Celegans with this two kind of demodulation methods respectively, compares their advantages and disadvantages and discusses their application condition.