Optical aberrations induced on fluorescence emission collected from biological specimens are a major limitation in modern fluorescence microscopy. Even for diffraction-limited commercial microscopes, the image quality is degraded when imaging inside a multicellular organism or in several layers of cells in tissue. Recent approaches have considered the use of adaptive optics to restore the image quality. For live specimen, one additional concern is the viability of the specimen. Light-Sheet Microscopy (LSM) is an innovative microscopy technology that allows high-resolution fast three-dimensional (3D) time-lapse imaging of biological specimens with reduced phototoxicity and photobleaching. As for other fluorescence microscopy techniques, LSM is subject to image degradation due to sample-induced optical aberrations. The goal of the proposed research is to develop a high-resolution custom-build Adaptive Optics Light-Sheet Microscope (AO-LSM) for 3D time-lapse (4D) biological imaging with compensation of sample generated optical aberration. Sample-induced aberrations have spatial variations in the volume of the thick specimen imaged and temporal variations because of the change in biological structures with time for live specimens. Our approach is divided in two specific aims. The first one will use samples with known and controlled aberrations to demonstrate light-sheet microscopy Adaptive Optics correction for static aberrations. The second specific aim will test innovative Adaptive Optics strategies and methods for the correction of dynamic optical aberrations (with temporal variation) during the embryonic development of a multicellular organism. The main significance of the project is the removal of the critical barrier of optical aberration in microscopy that occurs in thick sample (tissue, multicellular organisms). The proposed research will benefit a broad research community in biology and biomedical research. Impact will go beyond light-sheet microscopy, because the aberrations are also present for widely-used confocal and multiphoton microscopes. The project is innovative as it goes away from the current practice by correcting both the illumination and the emission (fluorescence) paths. The methodological approach starts from correcting known static aberrations to complex and dynamic aberrations in live specimen.

Public Health Relevance

The goal of this project is to develop a custom-build light-sheet microscope with adaptive optics to correct for the optical aberrations induced by live biological specimens. This will allow life scientists studying the structure and behavior of living systems to obtain an instrument with improved spatial resolution and obtain many more insights when studying cellular processes in physiologically relevant environment, outside of single cells cultured on coverslips. The proposed research is relevant to the public health because the instrument will benefit a broad research community in biology and biomedical research.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Academic Research Enhancement Awards (AREA) (R15)
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Special Emphasis Panel (ZRG1)
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Shabestari, Behrouz
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Delaware State University
Schools of Arts and Sciences
United States
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