Super Resolution Microscopy With Induced Optical Fluctuation

In conventinal microscopy, resolution is fundamentally limited by diffraction barrier. However, as phenomena within size of diffraction limit become important in many field, diffraction barrier becomes a big issue in conventional microscopy. To overcome this, many researchers have developed super-resolution methods using physical principles. Among them, lately, Super Resolution Optical fluctuation Imaging (SOFI) method developed by T. Dertinger et al, provides super-resolution image using statistical analysis of temporal optical fluctuation that comes from the blinking property of fluorophores. Because SOFI only requires opical fluctuation of fluorophores without any special equipment, it can be directly applied to conventional microscopy for super-resolution imaging. However, because intrinsic blinking property of fluorophores is uncontrollable, SOFI has practical challenges and limitations in some applications, including the limitation of available fluorophores and camera speed. To overcome these limitations, we proposed a new concept of SOFI using optical fluctuation induced by the illumination light with random patterns. Because the optical flcutuation does not come from intrinsic blinking property of fluorophore, but comes from the illumination light in this method, the blinking becomes a controllable property using external method. We employ speckle patterns as random patterns because speckle pattern has interesting statistical properties such as correlation. To verify the super-resolution capability of our proposed method, simple model simulation with only two flurophores was analytically solved using equations of SOFI and speckle pattern statistics. Also, to experimentally demonstrate it, diffuser and motorized stage were added in conventional microscopy to generate speckle patterns and statistical properties of speckle patterns. Using simulation and experiment, we theoretically and experimentally demonstrate the super-resolution imaging capability of our proposed method. Furthermore, proposed method gives super-resolution image of samples labelled with non-blinking fluorophores.