High-resolution in vivo imaging of mouse brain through the intact skull

Abstract

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...

Intact skull chronic windows for mesoscopic wide-field imaging in awake mice

“…A recent study demonstrated that advanced wavefront correction can compensate for high-order aberrations in the mouse skull, thus making it possible to resolve structures on the micron scale within the mouse brain (Park et al, 2015). Although we have not attempted two-photon imaging through our preparation, relatively minor additions to two-photon imaging hardware (a MEMS deformable mirror) may in the future make such experiments feasible.…”

In vivo volumetric imaging of biological dynamics in deep tissue via wavefront engineering

“…The highspeed operation is required in functional imaging with awake animals and the structural imaging of cellular dynamics, where the motion of the objectives-of-interest may introduce measurement artifacts. As we have demonstrated before [24][25][26]40], IMPACT works well in the ballistic regime for in vivo imaging, and the compensation wavefront can be effective for >60 minutes when imaging mouse cerebral cortex [17]. Further details of the two techniques can be found in our previous work [16,24].…”

“…Nevertheless, by combining IMPACT and the OPLUL based volumetric imaging, we can record the dynamics within the compensation volume in 3D with improved signal strength and signal-to-background-ratio, which provides a solution for volumetric imaging of biological dynamics at large depth. We expect that the deep tissue volumetric imaging demonstrated here can also be implemented by combining OPLUL with other configurations of wavefront correction, such as the single conjugated adaptive optics [40,[43][44][45][46] which has shown extended compensation field of view. Moreover, IMPACT and OPLUL are both compatible with fluorophores of longer excitation and emission wavelengths in two-photon and three-photon excitation [47], which can further improve the imaging depth.…”

Widefield fluorescence microscopy with sensor-based conjugate adaptive optics using oblique back illumination

“…Reports in both simulation [6][7][8] and experiment [9][10][11][12][13] have demonstrated the FOV advantage of conjugate AO as well as its feasibility. For example, conjugate AO has been applied in scanning microscopy configurations using both one- 9 and twophoton 10,11 microscopy. It has also been applied in widefield (i.e., nonscanning) microscopy configurations with transillumination geometries.…”