Abstract
Depth sensitive optical spectroscopy preferentially detects optical spectra from different depths in layered samples, which plays a crucial role in many applications such as the optical diagnosis of epithelial precancer and cancer. In depth sensitive optical measurements, multiple light scattering in tissues significantly degrades the depth sensitivity to a subsurface target layer. To address this issue, feedback based wavefront shaping led by guide stars can be used to refocus light to increase the depth sensitivity to a target layer. However, the lack of intrinsic guide stars in tissues or tissue-like samples often leads to poor enhancement in depth sensitive Raman/fluorescence measurements (~20% in the past literature) from the target layer due to the contribution from the overlaying non-target layer. In this study, we demonstrate that spatial filtering and spectral filtering can significantly improve the performance of depth sensitive fluorescence spectroscopy assisted by feedback based wavefront shaping in tissue-like scattering phantoms. The two filtering techniques work by effectively increasing the relative contribution from the target layer to the feedback signal during wavefront optimization through spatially and spectrally rejecting off-target fluorescence light, which is essentially similar to the role of time or coherence gating. When the filtering techniques are applied, a maximum of three-fold enhancement in fluorescence contribution from the target layer is observed, which is in contrast to nearly no enhancement in case of no filtering. This significant enhancement has not been reported previously for depth sensitive optical spectroscopy in the area of feedback based wavefront shaping. Therefore, our work represents a new advance towards the application of wavefront shaping in depth resolved optical spectroscopy for the characterization of layered structures such as epithelial tissues or drug tablets, in which the creation of an external guide star is challenging or not allowed.
Highlights
Depth dependent distribution of endogenous fluorophores and other intrinsic biomolecules reveals important diagnostic information about the progress of diseases such as cancer in epithelial tissues [1] is critical to early diagnosis
The positive role of spatial and spectral filtering in feedback based wavefront shaping can be attributed to the fact that both filtering techniques increase the relative contribution from the target layer to the feedback signal during wavefront optimization by spatially and spectrally rejecting off-target fluorescence light, which is similar to the roles of the pinhole and bandpass filter in confocal fluorescence microscopy
Spectral filtering can work for depth sensitive fluorescence spectroscopy in epithelial tissue since it is well known that endogenous fluorophores in the epithelium and stroma of epithelial tissues are different with distinctive peaks [36]
Summary
Depth dependent distribution of endogenous fluorophores and other intrinsic biomolecules reveals important diagnostic information about the progress of diseases such as cancer in epithelial tissues [1] is critical to early diagnosis. Optical spectroscopy [2], [3] are rugged and convenient for in vivo hand-held measurements but suffer from low spatial resolution due to the divergence nature of light coming out of optical fibers. Optical probes based on an objective lens [6] were proposed to implement non-contact measurements to reduce the uncertainty in contact pressure and spectral acquisition, which improves the spatial resolution since the light can be focused by the objective lens. This setup requires the movement of the objective
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