Optical coherence microscopy (OCM) provides non-invasive, label-free, cellular-resolution imaging based on optical scattering contrast. Its interferometric detection captures the optical field, providing opportunities for computational reconstruction. However, the depth coverage of OCM is restricted by defocus and photon collection, and its penetration depth is limited by multiple scattering (MS). Here, we propose integrating hardware and computational adaptive optics in different ways, to improve the throughput, penetration depth, and contrast of volumetric OCM. This hybrid adaptive optics (hyAO) approach splits the image formation process into a combination of hardware and computation components. For sparse sample imaging, we generated astigmatism using hardware adaptive optics (HAO) to achieve a more equalized photon distribution across depth, and removed the applied aberration (and defocus) via computational adaptive optics (CAO). We applied this hyAO method to perform 3D time-lapse imaging of in vitro fibroblast cell dynamics over a 1mm×1mm×1mm field-of-view with 2μm isotropic spatial resolution and 3-minute temporal resolution. The hyAO approach is not only beneficial for high-throughput volumetric imaging, but is also capable of suppressing MS/speckle. For scattering sample imaging, HAO was used to illuminate the sample volume with diverse aberrated point spread functions to decorrelate the MS/speckle fields, and CAO was applied to computationally mitigate the resolution penalty of these intentionally induced aberrations. By imaging with this aberration-diverse OCT using 12 volumetric reconstructions, we achieved a 10 dB enhancement in signal-to-background ratio at a USAF target plane beneath a scattering layer (7.2 scattering mean-free-path), and a 3× speckle contrast reduction within the scattering layer.
Optical coherence microscopy (OCM) is an interferometric imaging technique that enables high resolution, non-invasive imaging of 3D cell cultures and biological tissues. Volumetric imaging with OCM suffers a trade-off between high transverse resolution and poor depth-of-field resulting from defocus, optical aberrations, and reduced signal collection away from the focal plane. While defocus and aberrations can be compensated with computational methods such as interferometric synthetic aperture microscopy (ISAM) or computational adaptive optics (CAO), reduced signal collection must be physically addressed through optical hardware. Axial scanning of the focus is one approach, but comes at the cost of longer acquisition times, larger datasets, and greater image reconstruction times.
Given the capabilities of CAO to compensate for general phase aberrations, we present an alternative method to address the signal collection problem without axial scanning by using intentionally aberrated optical hardware. We demonstrate the use of an astigmatic spectral domain (SD-)OCM imaging system to enable single-acquisition volumetric OCM in 3D cell culture over an extended depth range, compared to a non-aberrated SD-OCM system. The transverse resolution of the non-aberrated and astigmatic imaging systems after application of CAO were 2 um and 2.2 um, respectively. The depth-range of effective signal collection about the nominal focal plane was increased from 100 um in the non-aberrated system to over 300 um in the astigmatic system, extending the range over which useful data may be acquired in a single OCM dataset. We anticipate that this method will enable high-throughput cellular-resolution imaging of dynamic biological systems over extended volumes.