Depth Limit of Imaging through Scattering Media using Selective Plane of Illumination Microscopy (SPIM)
Master of Science
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.
SPIM; depth limit; MFP; brain; scattering