Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

Marjolein B M Meddens; Sheng Liu; Patrick S Finnegan; Thayne L Edwards; Conrad D James; Keith A Lidke

doi:10.1364/BOE.7.002219

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

Biomed Opt Express. 2016 May 17;7(6):2219-36. doi: 10.1364/BOE.7.002219. eCollection 2016 Jun 1.

Authors

Marjolein B M Meddens¹, Sheng Liu², Patrick S Finnegan³, Thayne L Edwards³, Conrad D James³, Keith A Lidke⁴

Affiliations

¹ Department of Physics and Astronomy, University of New Mexico, 1919 Lomas Blvd NE, Albuquerque, NM 87131, USA; Department of Pathology, University of New Mexico, 2325 Camino de Salud, Albuquerque, NM 87131, USA.
² Department of Physics and Astronomy, University of New Mexico, 1919 Lomas Blvd NE, Albuquerque, NM 87131, USA; Current address: Weldon School of Biomedical Engineering, College of Engineering, Purdue University, West Lafayette, IN 47907, USA.
³ Sandia National Laboratories, 1515 Eubank SE, Albuquerque, NM 87123, USA.
⁴ Department of Physics and Astronomy, University of New Mexico, 1919 Lomas Blvd NE, Albuquerque, NM 87131, USA.

Abstract

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

Keywords: (100.6640) Superresolution; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (230.3990) Micro-optical devices.

Abstract

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