Three-dimensional Single-particle Tracking By Light Sheet Fluorescence Microscopy

Three-dimensional single-particle tracking(3D SPT)which can obtain realistic dynamics process information of bioparticels in living cells at nanometer and millisecond scale resolution has become an important research tool in the life sciences. However, 3D SPT poses several challenges in thick specimens, such as low signal to noise ratio, restricted imaging depth and poor spatial-temporal resolution. In this thesis, we present a 3D SPT strategy based on light sheet fluorescence microscopy combined with double-helix point spread function(DH-PSF) imaging to overcome these challenges. The main contributions of this thesis are summarized as follows:(1) The advantages and disadvantages of the existing SPT techniques are summarized and a novel 3D nanoscale SPT approach is developed that combines light sheet illumination technique with DH-PSF imaging for nanoscale axial localization.(2) A light sheet fluorescence microscopy was designed and constructed. The quantitative measurements of the characteristic parameters of the illumination light sheet(thickness2.24?m,depth-of-focus 26.3?m) are performed. Therefore, the results are consistent with the theoretical investigation.(3) The theories and technologies about double-helix point spread function are further illustrated. And the design and fabrication of a high-efficiency DH-PSF phase plate for 3D single-particle localization over an extended axial range are presented.(4) A completely new setup for 3D single-particle imaging are designed and constructed,that combines light sheet illumination technique with DH-PSF imaging for nanoscale axial localization. A direct comparison of this system and epi-illumination of model samples illustrate the clear image contrast and localization accuracy improvement of this system for thick samples.(5) The applications of the system to 3D SPT for single fluorescent bead in agarose solution are explored preliminarily. The experiments prove the capability of our setup for the study of fast and dynamic process in thick samples.Main innovation:(1) Created a simple optical sectioning microscope based on light sheet microscopy. The sample is illuminated perpendicular to the detection axis with a thin light sheet as thin as 2?m to overcome the limitation of high background noise of single-particle imaging.(2) Developed a 3D SPT strategy based on light sheet fluorescence microscopy combined with double-helix point spread function(DH-PSF) imaging to enhance the three dimensional localization accuracy and imaging depth of 3D SPT.