Label-free Far-field Super Resolution Microscopy Based On Illumination Modulation

Super resolution microscopy is an important tool for us to uncover this world.However,the techniques in the state of art are limited by the narrow field of view,the low imaging speed,the dependence on labelling,and the incapability of far-field visualization.The label-free far-field super resolution microscopy could most likely avoid the perturbance and damage to the sample during the imaging process,and shows great potential in many areas including biomedicine,physics,material science and so on.The researches demonstrated in this thesis have developed the label-free far-field super resolution microscopy techniques by illumination design.The major breakthroughs include:a)One dimensional nano-structure is firstly utilized for wide-field super resolution microscopy.We have realized the label-free far-field super resolution microscopy with a large field of view,which is two order of magnitude improved from the other reported label-free far-field super resolution microscopy techniques.Moreover,this method can be designed as a compact illumination module,giving super resolution capability to conventional microscopes.b)We have deeply studied the properties for the frequency shift microscopy,resorting to theoretical analysis,simulations,and experimental results.An image reconstruction method combined with a multiple illumination design for undistorted imaging was firstly proposed.c)A nonlinear ultraviolet photoacoustic microscope was developed to image the unlabelled cell nuclei with subdiffraction resolution for the first time.The structure of this thesis is demonstrated as follows.In the first Chapter,both the development of the microscopy and the typical label-free super resolution microscopes are introduced.In the second Chapter,a new design for evanescent illumination is proposed,which can upgrade the field of view and resolution for the label-free far-field super resolution microscopy.In the design,a fluorescent nanowire ring is combined with a film waveguide to achieve the evanescent illumination with high transverse wave vectors in a large area.Based on the frequency shift effect,the high spatial frequencies of the sample under such illumination can be extracted in the far field.The mechanisms and properties of this new design are studied systematically,and its versatility is demonstrated on various kinds of samples.In the third Chapter,the property of the frequency shift effect based imaging is analyzed in detail,and the nanowire ring shaped illumination technique is further optimized.Multiple wavelength illumination to broaden the far-field detectable spatial frequency range to achieve undistorted imaging is proposed,and the corresponding experimental methods as well as the algorithms are demonstrated.In the fourth Chapter,pump light and probe light are modulated in ultraviolet photoacoustic microscopy to induce nonlinear effect during the imaging process.The method can achieve sub-diffraction visualization for DNA/RNA and would find potential applications in clinical settings.In the last Chapter,all the work is summarized.