Enhanced 3D localization of individual RNA transcripts via astigmatic imaging

Evan P. Perillo; Leyma De Haro; Mary E. Phipps; Jennifer S. Martinez; Hsin-Chih Yeh; Andrew K. Dunn; Douglas P. Shepherd; James H. Werner

doi:10.1117/12.2038197

4 March 2014 Enhanced 3D localization of individual RNA transcripts via astigmatic imaging

Evan P. Perillo, Leyma De Haro, Mary E. Phipps, Jennifer S. Martinez, Hsin-Chih Yeh, Andrew K. Dunn, Douglas P. Shepherd, James H. Werner

Author Affiliations +

Proceedings Volume 8950, Single Molecule Spectroscopy and Superresolution Imaging VII; 895003 (2014) https://doi.org/10.1117/12.2038197
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Here we present an automated microscope capable of 3D multi-color single molecule localization of individual messenger RNA molecules in a wide range of cell types. We have implemented astigmatic imaging with a cylindrical lens to improve z-localization, and a maximum likelihood estimator on a graphics processing unit to improve localization precision and speed. This microscope will aid in gene expression analysis by its capability to perform high throughput imaging of thick cells and tissues while still maintaining sufficient z resolution to resolve single RNA transcripts in three dimensions. Enhanced z-localization allows for resolving membrane localized and co-localized transcripts.

Citation Download Citation

Evan P. Perillo, Leyma De Haro, Mary E. Phipps, Jennifer S. Martinez, Hsin-Chih Yeh, Andrew K. Dunn, Douglas P. Shepherd, and James H. Werner "Enhanced 3D localization of individual RNA transcripts via astigmatic imaging", Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 895003 (4 March 2014); https://doi.org/10.1117/12.2038197

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