Volumetric adaptive optical multiphoton microscope for in vivo imaging and photomanipulation

Abstract

Modern biological studies are mainly built upon observational sciences and thus relies heavily on imaging and manipulation tools. Light microscopy is one of the most widely recognized fundamental instrument in life science that can decipher biological processes over several length scales. Modern optical microscopes are combinations of laser engineering, optical design, software development and biochemical labelling. A well-orchestrated microscope should accumulate both high throughput information and also manipulate biological events of living cells within an animal in its native microenvironment. Multiphoton microscopy is widely used to study cellular and sub-cellular events ranging from immunological diseases model to neurological responses in living mice. A key success in the multiphoton microscope is the unprecedented imaging depth and imaging resolution from the nonlinear optical absorption. It is because of this nonlinear effect, fluorophores and specific proteins can be imaged and tracked over time. However, the inefficiencies of multiphoton microscopy during the delivering of a highly concentrated amount of photons over space and time within highly scattering tissue. In this thesis, I shall elaborate upon the design, development and application of a rapid volumetric scanning multiphoton microscope platform with adaptive optics capability for the intravital imaging and photomanipulation. The development of both the hardware and software was shown to a range of in vivo imaging experiments that especially focus on micro-vasculature imaging. A major milestone achievement is the experimental demonstration of video-rate adaptive optics capable of achieving 20 fps through raster aberration correction with time multiplexing. Overall, the final system achieves volumetric imaging (1~10 VPS) with an extendable imaging field of view (FOV) from 0.3 x 0.45 x 0.08 mm to 0.8 x 0.6 x 0.08 mm and a space bandwidth product (SBP- digital pixel density from 512 x 512 pixels ~ 10240 x 10240 pixels) in a single acquisition. These devices and functions are fully integrated into a customized control software named PScan, that provides not only an accessible operation interface for high throughput imaging but also offers photomanipulation capabilities. In the first chapter, I shall provide a general introduction and discussion of the development of fluorescence microscopy with the focus on multiphoton effect for deep tissue imaging and implementation of multiphoton microscopy. In the next three chapters, I shall describe our approach in designing and constructing the next generation of polygon based multiphoton system along with the control software PScan. Chapter 3 then illustrates our approach to achieve video-rate AO correction, named raster-scanning AO (RAO) that shows the capability of compensating for field-dependent aberration using time multiplexing with 10 ms update rate. Then, chapter 4 demonstrates the application of a piezo-deformable mirror to achieve volumetric imaging rates and a slit-based approach to achieve localized photomanipulation for fluorescence recovery after photobleaching (FRAP) and laser induced injury for in vivo thrombus studies. Finally, in chapter 5, I shall summaries the main features of the setup and also proposes several plans to further expanding the many functions of the microscope system for in vivo longitudinal photoconversion imaging. In summary, this thesis presents the detail of designing and building a state-of-art volumetric microscope that achieves multi-functionality imaging performance (speed, FOV, contrast, aberration-free) for in vivo studies of mice models. I anticipate that this volumetric microscope will be beneficial for a broader field of laser scanning microscopy for biologists and I believe that this thesis will impact various areas of biological sciences such as neurobiology, immunology and cancer research