Adaptive optics in nonlinear microscopy implemented with open-loop control and EMCCD-based Shack-Hartmann wavefront sensor

Nonlinear microscopy, with its unique advantages over conventional confocal fluorescence microscopy, has been widely adopted to study biological processes at the cellular level. However, like all other high-resolution optical imaging techniques, nonlinear microscopy suffers from focal degradation due to optical aberrations in the sample as a result of refractive index mismatch. Optical aberrations distort the wavefront of the excitation beam, causing the focal spot to be larger than the diffraction limit. Since the fluorescence efficiency scales nonlinearly with the profile of the focusing excitation beam, aberrations further degrade the image brightness in addition to resolution. In this dissertation I describe the design, characterization and experimentation of an adaptive optics (AO) nonlinear laser scanning microscope implemented with open-loop control and an EMCCD-based Shack-Hartmann wavefront sensor (EMCCD SHWFS) for aberration compensation. Adaptive optics (AO), originally designed for ground-based astronomical observatories to correct for the aberrations from atmospheric turbulence while imaging distant stars and planets, has benefited many biomedical imaging platforms. We integrated a microelectromechanical system (MEMS) deformable mirror (DM) into our nonlinear laser scanning microscope. With an accurate open-loop control mechanism, which predicts the control voltages and generates a prescribed surface shape on the MEMS DM, known aberrations in the system can be compensated for with this computationally simple and inherently fast method. The use of a nonlinear guide star imbedded within the sample can reflect the sample aberration. However, the low level of nonlinear fluorescence signal is usually detected by photomultiplier tubes (PMT) and is below the sensitivity of a conventional charge-coupled device (CCD) based Shack-Hartmann wavefront sensor. This dissertation also describes the design of an EMCCD SHWFS to measure the wavefront distortion from the nonlinear guide star and aberration compensation from the skull bone marrow of a live mouse is demonstrated using the described system