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Optica. 2016 Aug 20;3(8):897-910. Epub 2016 Aug 11.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.

Author information

1
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
2
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
3
Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
4
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA; State Key Lab of Modern Optical Instrumentation, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310027, China.
5
Department of Biology, Indiana University Bloomington, Bloomington, Indiana 47405, USA.
6
Department of Radiology, University of Chicago, Chicago, Illinois 60637, USA.
7
State Key Lab of Modern Optical Instrumentation, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310027, China.
8
Department of Radiology, University of Chicago, Chicago, Illinois 60637, USA; Whitman Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
9
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA; Whitman Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.

Abstract

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.

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