Deep-tissue super-resolved microscopy with multiphoton spatial frequency-modulated imaging (MP-SPIFI)

Colorado State University, Fort Collins CO

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Abstract

Multiphoton (MP) optical microscopy is a highly successful method for optical imaging deep in tissues. MP microscopy forms high quality images deeper than other methods because the images are formed with absorption or coherent scattering that displays a nonlinear dependence on optical intensity. The nonlinear excitation means that the light used is longer in wavelength than many imaging methods ? and is degraded much less by optical scattering as it propagates into tissue. The nonlinear excitation also allows light generation to be primarily confined to a small excitation volume. The localization of the excitation to near the focal volume reduces photobleaching and background light generation outside of the focal region. In addition, the all of the light generated by the nonlinear interaction ? including light multiply scattered by the tissue ? can be collected as it exits the tissue and the brightness is assigned to the known focal position to form an image. Despite the many benefits, MP optical microscopy suffers a critical drawback: to image deep in tissue, the numerical aperture of the objective lens used to focus the light into the tissue must be low enough for the working distance to be sufficiently long to allow the focus to be placed deep into the tissue. This low numerical aperture limits the spatial resolution of MP microscopy, and thus limits the spatial structures that can be resolved deep into tissues to slightly below a micron.In this proposal, we describe a new microscope that will remedy this situation by enabling super-resolution imaging deep in optically scattering tissue (> 1mm deep) for the first time. While super-resolution imaging has revolutionized biological imaging, current methods do not work in tissues ? preventing their use in in vivo imaging of organisms. Our new approach is the only super- resolution imaging method that records images with coherent nonlinear scattered light and light emitted after nonlinear absorption. The proposed microscope builds on our recent demonstration of the first super-resolution imaging method that can record super-resolved images using both nonlinear optical absorption and coherent nonlinear optical scattering. In our approach, a femtosecond laser pulse is brought to a line focus and modulated in space and time. Light emitted following nonlinear optical absorption or light generated by coherent nonlinear scattering is collected on a single pixel detector. Nonlinear excitation of the modulated light enables images with finer spatial resolution than conventional MP microscopy. The ability to image deep into scattering objects will be first tested by carefully prepared tissue simulating phantoms to quantify the depth penetration and spatial resolution. After validating the approach, the first SR images will be captured in fixed thick tissue slices. The resolution enhancements improve commensurately with increased nonlinear interaction order. The development of this new imaging technology will enable unprecedented high spatial resolution imaging deep in live animals.