Optimization of a 3D Wide-field Super-resolutionOptical Sectioning Microscope

• Main Authors: Ssu-hsuan Chou, 周思瑄
• Other Authors: Jiunn-Yuan Lin
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/86815363720654916922

碩士 === 國立中正大學 === 物理所 === 98 === Wide-field optical microscopy is one of the most popular techniques for observing biological specimens. However, the optical resolution of microscopy is limited to be larger than ~ 0.5??laterally and 1.5? in depth due to diffraction effect. It is shown that the wide-field super-resolution imaging with the lateral resolution on the order of 100 nm is achievable if the sample is illuminated with periodic structured light. In order to retrieve a super-resolution image, several images illuminated with space-shifted patterned light must be taken and processed numerically. This will greatly reduce the imaging frame rate as mechanical movements are involved. In this thesis, we show that optically sectioned super-resolution microscopic imaging can be achieved by using a single SLM to modulate the illumination light. The 2D sinusoidal modulation mesh pattern projected on the sample is shifted by rapidly varying the driving signal on the LCoS panel at a frame-refresh rate as high as 60 Hz. The sectioned and super-resolved image is generated simultaneously from the same set of patterned excitation images, and the image acquisition rate of the image set can be as high as one set per second. By varying the period of 2D sinusoidal pattern on SLM, we can control the enhancement factor of the spatial resolution while keeping the field of view of the image fixed. With the SLM based super-resolution sectioning microscope, we improve the lateral resolution to 0.25 wavelengths and achieve a depth resolution of 0.38 wavelengths simultaneously. By taking the advantage of our advanced imaging system, 3D sectioning images of BPAE cells with mouse anti-?-tubulin and f-actin have been successfully acquired. These results demonstrate the ability of observing the sub-cell structure. Furthermore, this simple and high-resolution wide-field optical microscopy can be easily implemented on conventional fluorescence microscopes and has the potential to be applied to dynamical analyses of the organelles inside a live cell.