Volume reconstruction and resolution limits for three dimensional snapshot microscopy
- Three-dimensional snapshot microscopy refers to any technique capable of performing volumetric imaging of microscopic samples using information captured in a single photographic exposure. Unlike scanning microscopes, which collect volumetric information over time, 3-D snapshot microscopes can capture volumes at speeds limited only by the frame rate of the image sensor. Synchronous imaging is made possible by encoding depth information in the shape of the microscope's point response function (PRF). Each position in the volume produces a different, distinctive light intensity pattern on the camera sensor, and these patterns can be recognized by a computer algorithm and used to computationally reconstruct a full volume. In this work we explore two different 3-D snapshot microscopes for imaging of weakly scattering, fluorescent specimens. The first, the light field microscope, employs an array of microlenses to decompose light into different angular projections of the volume in a manner similar to computed tomography. We present an optical model for light field microscopy based on wave optics and a 3-D reconstruction method which we solve using a GPU-accelerated iterative algorithm. Theoretical resolution limits for the light field microscope are discussed and compared with experimental measurements using a USAF 1951 resolution target, pollen grains, and fluorescent beads. We also summarize our application of light field microscopy in neuroscience where we have used it to perform 3-D calcium imaging. Using this technique, we have recorded the activity of thousands of neurons in the brains of awake, behaving animals. Our second approach to 3-D snapshot imaging uses phase masks, rather than a microlens array, to encode volumetric information. We have designed a ``helical focus'' phase mask that generates a PRF that rotates as the microscope is defocused. This PRF contains a single lobe that does not change in size or shape as it rotates, thereby enabling imaging with consistent resolution over a configurable depth range of up to hundreds of micrometers. Further, we propose a design for a 3-D snapshot microscope that uses two such masks (in different light paths, but with simultaneous acquisition using two frame synchronized cameras) to capture volumetric information. Our optical simulations suggest that this microscope is capable of performing 3-D imaging at resolutions exceeding that of light field microscopy when imaging sparse volumes. We show these results and compare the two techniques.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Computer Science Department.
|Statement of responsibility
|Submitted to the Department of Computer Science.
|Thesis (Ph.D.)--Stanford University, 2017.
- © 2017 by Michael Joseph Broxton
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