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  • Deep Imaging in Scattering Media with Single Photon Selective Plane

    Illumination Microscopy (SPIM)

    Adithya Kumar Pediredla1,∗, Shizheng Zhang1,∗, Ben Avants1, Fan Ye1, Shin Nagayama2, Ziying Chen1, Caleb Kemere1,3, Jacob

    Robinson1,3,4,†, and Ashok Veeraraghavan1,5,‡ 1 Department of Electrical and Computer Engineering, Rice University, Houston, TX 77005,

    USA 2 Department of Neurobiology and Anatomy, University of Texas, Medical school at Houston,

    TX 77030, USA 3 Department of Bioengineering, Rice University, Houston, TX 77005, USA

    4 Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA 5 Department of Computer Science, Rice University, Houston, TX 77005, USA

    ∗ Both authors contributed equally to the work † jtrobinson@rice.edu

    ‡vashok@rice.edu

    Abstract: In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. In this paper, using a combination of theoretical and experimental techniques, we show that Selective Plane Illumination Microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (approximately 10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations where 2PM systems would be too large or costly.

    © 2016 Optical Society of America OCIS codes: (030.5620) Radiative transfer; (110.0110) Imaging systems; (180.2520) Fluores- cence microscopy; (180.6900) Three-dimensional microscopy; (290.0290) Scattering; Selec- tive plane illumination microscopy;

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  • Fig. 1. SPIM Experimental Configuration. The excitation direction and the fluorescence detection direction are orthogonal to each other. The illumination optics are designed to create a thin light sheet at the focal plane of the imaging objective. By moving the sample in z-direction, one can reconstruct a 3D image of the sample. The right side of the figure shows images at three different depths (z). The quality of image reduces as one images deeper from the sample surface. As a result, there is a limit beyond which the informa- tion in the image is completely lost. In this paper, we characterize both theoretically and experimentally this imaging depth limit for SPIM.

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