Multiphoton microscopy is the current method of choice for in vivo deep-tissue imaging. The long laser wavelength suffers less scattering, and the 3D-confined excitation permits the use of scattered signal light. However, the imaging depth is still limited because of the complex refractive index distribution of biological tissue, which scrambles the incident light and destroys the optical focus needed for high resolution imaging. Here, we demonstrate a wavefrontshaping scheme that allows clear imaging through extremely turbid biological tissue, such as the skull, over an extended corrected field of view (FOV). The complex wavefront correction is obtained and directly conjugated to the turbid layer in a noninvasive manner. Using this technique, we demonstrate in vivo submicron-resolution imaging of neural dendrites and microglia dynamics through the intact skulls of adult mice. This is the first observation, to our knowledge, of dynamic morphological changes of microglia through the intact skull, allowing truly noninvasive studies of microglial immune activities free from external perturbations.neuroimaging | nonlinear microscopy | wavefront shaping | immunology | adaptive optics B reakthroughs in imaging technologies have been one of the major driving forces of new discoveries in biology (1). In the past two decades, we have witnessed that the advances of superresolution microscopy revolutionized our understanding of the complex dynamics in single cells (2). However, the techniques that work well on cultured cells often fail when being used to observe the cells in their native environment inside a living organism, the most ideal condition for biological studies. These difficulties result from the optical wavefront distortions induced by the inhomogeneous refractive index distribution in biological tissue.Because of this limitation, imaging inside deep tissue has been a challenging task, and the common goal of state of the art deep tissue-imaging methods is to recover the diffraction-limited resolution. These efforts can be categorized largely into two groups: the first capitalizes the use of longer wavelengths (3, 4) that undergo less scattering, whereas the second controls the optical wavefront, so that the wavefront distortions induced by the sample can be compensated (5-30). Longer wavelength excitation has demonstrated impressive penetration depths in the exposed brain (3) and imaging of the brain vascular structures beneath the skull (4). However, at the current state, using longer wavelengths is not a simple option because it requires special illumination sources [e.g., low repetition rate, high energy pulses (3)] or fluorescent probes (4), and provides only moderate spatial resolutions. In comparison, wavefront shaping allows the use of conventional light sources as well as the wide selection of wellestablished labels and functional indicators (31).Previous exciting works in adaptive optics (AO) have exploited these advantages and demonstrated aberration correction for a large field of view (FOV) by avera...

For in vivo deep imaging at high spatiotemporal resolutions, we developed Multi-Pupil Adaptive Optics (MPAO) which enables simultaneous wavefront correction over a large imaging field-of-view. The current implementation improves correction area by nine times over that of conventional methods. MPAO’s capability of spatially independent wavefront control further enables 3D nonplanar imaging. We applied MPAO to in vivo structural and functional imaging of biological dynamics in mammalian brain.

Despite the unique advantages of optical microscopy for molecular specific high resolution imaging of living structure in both space and time, current applications are mostly limited to research settings. This is due to the aberrations and multiple scattering that is induced by the inhomogeneous refractive boundaries that are inherent to biological systems. However, recent developments in adaptive optics and wavefront shaping have shown that high resolution optical imaging is not fundamentally limited only to the observation of single cells, but can be significantly enhanced to realize deep tissue imaging. To provide insight into how these two closely related fields can expand the limits of bio imaging, we review the recent progresses in their performance and applicable range of studies as well as potential future research directions to push the limits of deep tissue imaging.

Focusing through scattering media is a subject of great interest due to its direct impact in the field of biomedical optics. However, the greatest barrier currently limiting direct applications is the fact that most scattering media that we wish to deliver light through are dynamic. To focus or deliver light through dynamic scattering media, using a digital micromirror device (DMD) has been demonstrated to be a potential solution, as it enables fast modulation speeds. However, since a DMD is a binary amplitude modulator, the large number of controlled modes needed to acquire adequate focus enhancement has limited optimal usage. Here we demonstrate a novel (to the best of our knowledge) scheme to use the “thrown-away” components of light to effectively use a binary amplitude DMD as a binary phase modulator, thereby increasing the correction efficiency by a factor of two. Our concept can be applied to any iterative optimization algorithm and can speed up the iterative optimization process by increasing the enhancement factor, rather than the measurement or modulation speeds.