COMBINING WIDE-FIELD AND SCANNING LASER MICROSCOPY FOR FAST 3D IMAGING
National University of Singapore, Singapore
A wide range of light microscopy methods have found their applications in various biomedical researches. Different microscopy techniques have different advantages and disadvantages. For example, widefield optical microscopy has the advantages of being convenient and high imaging speed. It lacks the depth sectioning capability, however, as axial high-frequency components in its 3D optical transfer function is missing. Therefore, it is impossible to use a wide-field microscope to obtain 3D structures of a specimen. Laser scanning microscopy method, such as confocal microscopy can achieve excellent optical sectioning as well as high axial resolution. While conventional confocal microscopy is a standard tool for 3D visualization of fixed samples, their long image acquisition time and excessive photobleaching make it less suitable for real-time in vivo imaging of live samples. We have developed a novel method to effectively combine the information from both scanning microscopy and wide-field microscopy imaging modes. A prototype hybrid microscope has been designed and implemented. We have verified imaging performances of the hybrid microscope with imaging results acquired from various live samples. The reduced photobleaching, improved imaging speed, and improved optical sectioning have been demonstrated.