Infrared Photothermal Spectroscopy And Raman Spectroscopy Imaging Microscopy And Its Applications For Single Cell

Vibrational spectroscopic imaging microscopy is a cutting-edge technique in the field of biomedicine,which can non-invasively identify the molecular information of a single cell structure and visually analyze its chemical composition,providing more chances for the early stage diseases diagnosis and drug treatment.The traditional infrared spectroscopic imaging microscopy has the advantages of large absorption cross-section and fast imaging speed.However,due to the poor spatial resolution and the disturbance of strong water absorption background,the traditional infrared spectroscopic imaging microscopy is unsuitable for research in active biological samples.Infrared photothermal spectroscopic imaging microscopy is an emerging label-free microscopic imaging technology,which indirectly detects the variation in far field distribution or intensity of another probe light caused by the photoinduced refractive index change influenced by infrared absorption.In addition to sub-micron lateral spatial resolution and micron-level axial resolution,this technique is insensitive to the background disturbance caused by water absorption,which is capable of live cell imaging.Compared with infrared spectroscopic imaging microscopy,Raman spectroscopic imaging microscopy can reflect different molecular structure information.It is suitable for studying active biological samples because of the merits of high spatial resolution and insensitive to water disturbance.However,the slow imaging speed of Raman spectroscopy imaging microscopy limits its further development.Due to the difference in generation mechanism,the infrared spectroscopy and Raman spectroscopy reflect the complementary information of molecular structure,which enables them to provide much comprehensive analysis for biological samples.On the basis,this thesis combines infrared photothermal spectroscopic imaging microscopy and Raman spectroscopy,and proposes a new Raman integrated infrared photothermal imaging microscope(IRaman).Infrared photothermal images are employed to guide the acquisition of infrared spectroscopy and Raman spectroscopy.Thus,IRaman takes full advangages of the complementary characteristics of infrared photothermal spectroscopy microscopic imaging and Raman spectroscopy to carry out the research on living biological cells.This thesis is conducted by the following five parts:1.The theory of infrared spectroscopy and Raman spectroscopy is investigated,and then the scheme of connecting Raman spectroscopy on the infrared photothermal spectroscopic imaging microscopy is proposed.The optical principles and complementary characteristics of infrared photothermal spectroscopy and Raman spectroscopy are compared.The feasibility analysis of the combination of the two technologies is carried out,and the signal generation principle,noise sources and spatial resolution of IRaman are studied.2.The instrument design and system construction of IRaman setup are completed.The design of the counter-propagating optical path improves the spatial resolution of the system;the infrared photothermal image is used to guide the collection of infrared photothermal spectrum and Raman spectrum,and the simultaneous acquisition of infrared spectrum and Raman spectrum of the same sample at the same position is realized.3.The calibration experiments and the performance tests of IRaman setup are completed.In the experiments,the 500 nm polymethyl methacrylate microspheres and dimethyl sulfoxide organic solvent are selected as standard samples.Then,calibration experiments and performance tests were conducted in the infrared photothermal imaging part and the Raman spectral part,respectively.Experiments results proved that the IRaman setup has the advantages of fast imaging speed,high spatial resolution and fast spectral acquisition speed.4.The medical application researches on living biological cells are carried out.In this part,the analysis of the localization and saturation of lipids in adipocyte,the localization and analysis of chemical components in cancer cell,as well as the imaging of single cell distribution and chemical components of bacteria are presented.5.The researches on the changes of internal chemical composition of bacteria after drug treatment are presented.Combined with the principal component analysis algorithm,the bacterial spectrum was visually analyzed and successfully distinguished between the bacteria treated by the drug and the bacteria not treated by the drug.In conclusion,as a new system to combine infrared photothermal spectroscopy and Raman spectroscopy,IRaman setup has high spatial resolution and high signal-to-noise ratio,which can quickly obtain chemical imaging and spectral information of biological single cell structure.IRaman setup shows the great application potential in the early stage of disease diagnosis and drug treatment.