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Biomed Opt Express. 2016 Jan 14;7(2):454-66. doi: 10.1364/BOE.7.000454. eCollection 2016 Feb 1.

Assessing the imaging performance of light sheet microscopies in highly scattering tissues.

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1
Department of Mechanical Engineering, University of Washington, Seattle, WA, USA.

Abstract

Light sheet microscopy (LSM) has emerged as an optical-imaging method for high spatiotemporal volumetric imaging of relatively transparent samples. While this capability has allowed the technique to be highly impactful in fields such as developmental biology, applications involving highly scattering thick tissues have been largely unexplored. Herein, we employ Monte Carlo simulations to explore the use of LSM for imaging turbid media. In particular, due to its similarity to dual-axis confocal (DAC) microscopy, we compare LSM performance to point-scanned (PS-DAC) and line-scanned (LS-DAC) dual-axis confocal microscopy techniques that have been previously shown to produce high-quality images at round-trip optical lengths of ~9 - 10 and ~3 - 4 respectively. The results of this study indicate that LSM using widefield collection (WF-LSM) provides comparable performance to LS-DAC in thick tissues, due to the fact that they both utilize an illumination beam focused in one dimension (i.e. a line or sheet). On the other hand, LSM using confocal line detection (CL-LSM) is more analogous to PS-DAC microscopy, in which the illumination beam is focused in two dimensions to a point. The imaging depth of LSM is only slightly inferior to DAC (~2 - 3 and ~6 - 7 optical lengths for WF-LSM and CL-LSM respectively) due to the use of a lower numerical aperture (NA) illumination beam for extended imaging along the illumination axis. Therefore, we conclude that the ability to image deeply is dictated most by the confocality of the microscope technique. In addition, we find that imaging resolution is mostly dependent on the collection NA, and is relatively invariant to imaging depth in a homogeneous scattering medium. Our results indicate that superficial imaging of highly scattering tissues using light sheet microscopy is possible.

KEYWORDS:

(110.0113) Imaging through turbid media; (170.1790) Confocal microscopy; (170.2520) Fluorescence microscopy; (170.3880) Medical and biological imaging; (170.5810) Scanning microscopy

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