Deep and Fast High-Resolution 3D Microscopy
Abstract
Acquisition speed in high-resolution imaging in three dimensions (3D) remainsa major challenge in modern microscopy. When 3D information is collected sequentially, acquisition speed is limited and increases with the sample volume size imaged. Aberration-corrected multifocus microscopy (MFM) employs diffractive Fourier optics to multiplex and refocus the microscope image to enable truly simultaneous 3D imaging, without loss of resolution. This thesis describes a new type of ultra-fast 25-camera-array multifocus microscope (M25) which employs an array of small, fast CMOS cameras sensitive enough for live 3D fluorescence imaging. The use of multiple sensors allows M25 to significantly push acquisition speed, depth or field, and field of view to capture 130×130×50 μm3 volumes at ~100 Hz. This new optical design also employs a radically simplified chromatic correction module from classical MFM systems, consisting of simple pairs of diffractive elements. The prototype instrument here described is customized for functional neural circuit imaging and locomotion in small model organisms and is demonstrated on Caenorhabditis elegans, fruit fly (Drosophila melanogaster), and lamprey (Petromyzon marinus).