Two-photon Excitation Laser Scanning Stereomicroscopy

Two-photon excitation fluorescence microscopy is widely used in the research fields,such as physiology,neurobiology,embryology and tissue engineering,due to its three dimensional(3D)imaging capability and deep penetration depth.However,in most of the current two-photon excitation fluorescence microscopes,3D images are acquired by using two dimensional lateral pointwise scanning and relative slow mechanical z-scanning,inasmuch the imaging speed is too slow to capture rapid physiological and functional processes.In this thesis,a two-photon excitation laser scanning stereomicroscopy has been implemented,taking advantages of both stereo vision and extended depth of field microscopy.This new technology improves the3 D imaging speed over ten times and enables real time volumetric imaging for biomedical research.The main contributions of this thesis work include:1.A stereo-scanner,a key component of the laser scanning stereomicroscopy,has been invented to switch views rapidly.Based on the basic principles of laser scanning,the stereo-scanner can scan the extended focus with three degrees of freedom: two dimensions for lateral scanning and an additional dimension for beam tilting.The scanner hardware and control software has been developed to allow switching between the two views within a millisecond.The problem of simultaneity of two imaging pathway is solved by this design.2.Theoretical analysis and numerical modeling of the stereomicroscopy has been performed.The image formation in two-photon excitation laser scanning stereomicroscopy has been investigated both theoretically and experimentally.A complete model of the microscope system including every optical element has been presented in Fourier optics or Rayleigh-Sommerfeld diffraction integral,providing more realistic simulation than the optical ray tracing method.Bessel beams created with different axicons are analyzed considering the influence of the round tip of the axicon.The imaging performance in frequency domain is analyzed based on the modeling of extended focus.3.A two-photon excitation laser scanning stereomicroscope with real-time stereoscopic display system has been built;the system consists of a ultrafast laser system,the stereo-scanner,a relay system,a specimen stage,and a signal acquisition and real-time display system.The corresponding hardware control program and image acquisition and processing program have been written.An extended focus measuring system has been developed with the help of a moving reflective mirror.A module for 3D display with shutter glasses has been developed,allowing for real-time observation of dynamic samples.The whole stereomicroscope can image a three dimensional volume at 1.48 Hz with an image size of 512 by 512 pixels;the lateral resolution is about 700 nm with an axicon of 1.15°.4.Depth recovering methods for fluorescence stereo images and three-dimensional reconstruction methods have been developed.Based on the epipolar constraint geometry of the stereomicroscopy,the depth information was recovered by using feature-based correspondence algorithm and modified normalized cross correlation algorithm for sparse samples and dense samples,respectively.A method for depth recovering and three-dimensional reconstruction for moving objects has been successfully developed.