High speed 3D two-photon fluorescence microscopy by femtosecond laser pulses

A high speed two-photon fluorescence microscopy system based on 2D galvanometer scanning is developed, and imaging of Rhodamine B samples and labeled Caski cells is performed. Coherent Micra-5 femtosecond laser is used as the light source, which has 82 MHz frequency, 45 fs pulse width and 400 mW average power. Galvo mirrors and prism pairs are applied in order to obtain higher imaging speed and better imaging resolution respectively. To prove the ability of system, two-photon imaging of Rhodamine B samples and Caski cells labeled by Rhodamine-dyed phalloidin are performed. The results show that the system imaging speed is greatly improved from previous work. The acquisition time reaches the order of 1frame/s, which makes up imprecision of mechanical translation platform and reduces photobleaching and structure damage. By measuring FWHM of lined region of image, lateral resolution is confirmed to be 1.05μm; the vertical resolution of the system can reach 3μm, which is restricted by Z-axis step motor. What's more, images of different frames are reconstructed to perform three-dimensional imaging. The ability of the phalloidin's specific binding to the cell is also verified by observing obtained two-photon image.