Browsing by Author "Zhang, Shizheng"

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    Deep imaging in scattering media with selective plane illumination microscopy
    (SPIE, 2016) Pediredla, Adithya Kumar; Zhang, Shizheng; Avants, Ben; Ye, Fan; Nagayama, Shin; Chen, Ziying; Kemere, Caleb; Robinson, Jacob T.; Veeraraghavan, Ashok
    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. Using a combination of theoretical and experimental techniques, we show that if the excitation path length can be minimized, 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 (∼10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations in which 2PM systems would be too large or costly.
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    Depth Limit of Imaging through Scattering Media using Selective Plane of Illumination Microscopy (SPIM)
    (2015-12-03) Zhang, Shizheng; Veeraraghavan, Ashok; Robinson, Jacob T; Kemere, Caleb
    In most biological tissues, the maximum optical imaging depth is limited by light scattering. Confocal and multi-photon microscopy have been developed to increase the imaging depth by limiting the amount of scattered light that reaches the detector, however, these techniques acquire images one point at a time resulting in reduced image acquisition speed. Recently, Selective Plane of Illumination Microscopy (SPIM) has emerged as an alternative 3D microscopy technique with faster image acquisition speeds, enabled by capturing entire 2D planes rather than individual points. While the advantages of SPIM for high speed imaging are understood, here we demonstrate that SPIM also increases the imaging depth in scattering media compared to confocal and epifluorescence techniques. We show both analytically and experimentally that SPIM can image 2-3 times deeper than confocal microscopy (~10x the mean scattering length). The primary reason for the deeper imaging capability of SPIM is the fact that off-axis illumination reduces the out-of-focus fluorescence above the imaging plane. We find that for scattering media, multi- photon microscopy can image deeper than SPIM; however, the fact that SPIM does not require a high-power pulsed laser makes this approach a lower cost alternative to multi-photon microscopy for imaging into scattering media beyond the depths of conventional single photon microscopy techniques.