Abstract
We report an innovative technique for the visualization of cells through an overlying scattering medium by combining femtosecond laser bone ablation and two-photon excitation fluorescence (TPEF) microscopy. We demonstrate the technique by imaging hair cells in an intact mouse cochlea ex vivo. Intracochlear imaging is important for the assessment of hearing disorders. However, the small size of the cochlea and its encasement in the densest bone in the body present challenging obstacles, preventing the visualization of the intracochlear microanatomy using standard clinical imaging modalities. The controlled laser ablation reduces the optical scattering of the cochlear bone while the TPEF allows visualization of individual cells behind the bone. We implemented optical coherence tomography (OCT) simultaneously with the laser ablation to enhance the precision of the ablation and prevent inadvertent damage to the cells behind the bone.
Highlights
Seeing through scattering media is one of the major challenges for optical imaging
We explore an alternative method that involves the creation of artificial windows in scattering media by thinning them down to a thickness approximately equal to one photon mean free path (MFP) using femtosecond laser ablation
We investigated the effects of pulse energy, and fluence, on the bone ablation while maintaining a pulse width of 330 fs and a repetition rate of 5 kHz throughout this work
Summary
An emerging class of wave front shaping techniques undoes the distortion due to scattering through a carefully shaped wave front [1,2,3] By sending such a shaped wave front back through the same medium, the scattering can be unscrambled and the original incident wave front is recovered. This can be used to produce a laser focus behind the scattering medium [4,5], which can be scanned for imaging via the correlation in the scattered wave front (memory effect) [6]. Great challenges remain for obtaining access from both sides of the scattering layer [9], or placing proper beacons in the target region [1]
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