Temporal Focusing-Based Multiphoton Excitation Microscopy

摘要

A microscope based on temporal focusing offering widefield multiphoton excitation has been developed to provide fast three-dimensional (3D) biomedical imaging and microprocessing. This configuration can produce multiphoton images at a frame rate greater than 100 Hz with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Also, three techniques have been adopted to promote the spatial resolution. Furthermore, by replacing the conventional diffraction grating with a digital micromirror device, the setup can provide temporal focusing and arbitrary pattern illumination simultaneously. In the spatial resolution improvement, the lateral and axial resolutions are enhanced to 168 nm and 1.22 μm by using a second order nonlinear structured-illumination microscopy. Then, the lateral resolution is further improved to 50 nm with the fast frame rate by integrating an astigmatism approach. Finally, a wavefront sensorless adaptive optics system was utilized to correct the disturbances induced from the sample and the environment. In the biomedical applications, the system provides fast 3D biomedical molecular imaging, but additionally, it also achieves freeform 3D protein microstructures and multiphoton-induced ablation.