Three-dimensional focusing through scattering media using conjugate adaptive optics with remote focusing (CAORF).

The small correction volume for conventional wavefront shaping methods limits their application in biological imaging through scattering media. We demonstrate large volume wavefront shaping through a scattering layer with a single correction by conjugate adaptive optics and remote focusing (CAORF). The remote focusing module can maintain the conjugation between the adaptive optical (AO) element and the scattering layer during three-dimensional scanning. This new configuration provides a wider correction volume by better utilization of the memory effect in a fast three-dimensional laser scanning microscope. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We also demonstrate three-dimensional fluorescence imaging, multi-spot patterning through a scattering layer and two-photon imaging through mouse skull tissue.

Enhancing image quality in cleared tissue with adaptive optics.

Our ability to see fine detail at depth in tissues is limited by scattering and other refractive characteristics of the tissue. For fixed tissue, we can limit scattering with a variety of clearing protocols. This allows us to see deeper but not necessarily clearer. Refractive aberrations caused by the bulk index of refraction of the tissue and its variations continue to limit our ability to see fine detail. Refractive aberrations are made up of spherical and other Zernike modes, which can be significant at depth. Spherical aberration that is common across the imaging field can be corrected using an objective correcting collar, although this can require manual intervention. Other aberrations may vary across the imaging field and can only be effectively corrected using adaptive optics. Adaptive optics can also correct other aberrations simultaneously with the spherical aberration, eliminating manual intervention and speeding imaging. We use an adaptive optics two-photon microscope to examine the impact of the spherical and higher order aberrations on imaging and contrast the effect of compensating only for spherical aberration against compensating for the first 22 Zernike aberrations in two tissue types. Increase in image intensity by 1.6× and reduction of root mean square error by 3× are demonstrated.