Abstract
BackgroundIf structures of interest are hidden beneath turbid layers such as biological tissues, imaging becomes challenging, even impossible. However, if the point spread function of the system is known from the presence of a guide star, application of common deconvolution algorithms can be a convenient approach to reconstruct even heavily scrambled images. In this work, we present the severity of scattering and the capability of deconvolution techniques in optical settings realistically mimicking biological applications.MethodsWe determine the point spread function (PSF) of the optical path using a single fluorescent bead hidden behind a scattering layer. Once the PSF is obtained, a scene containing several beads is brought to the exact the same position behind the scattering layer. The scrambled image of the scene is then deconvoluted with the PSF. Plastic films and thin slices of chicken tissues are used as scattering layers.ResultsDespite the low signal provided by small fluorescent particles and their short distance of a few millimeters to the turbid media, the reconstructed images reproduced the original scenes successfully. The spatial variance of the PSF caused by the inhomogeneous scattering layer mainly limited the size of the reconstructed area.ConclusionOur method overcomes the negative effects of scattering on the detection side of an imaging system. However, it can be combined with wavefront shaping methods optimizing the illumination path as well leading to even further increase of signal to noise ratio and image quality. The required guide star can be brought inside the biological sample to a desired position using optical fibers as a light guide or using capillaries filled with bright fluorescent molecules.
Highlights
If structures of interest are hidden beneath turbid layers such as biological tissues, imaging becomes challenging, even impossible
The required guide star can be brought inside the biological sample to a desired position using optical fibers as a light guide or using capillaries filled with bright fluorescent molecules
The structures of interest in biological tissues are covered with scattering layers both on the illumination path and detection path
Summary
If structures of interest are hidden beneath turbid layers such as biological tissues, imaging becomes challenging, even impossible. Traditional adaptive optics techniques in fluorescence microscopy rely on direct wavefront measurements, employing e.g. a Hartmann-Shack sensor [1,2,3]. This sensor detects and analyses either the aberrant wavefront of the backscattered excitation light [4, 5] or the wavefront of a fluorescent guide star [6,7,8]. The compensation of the aberrations is performed by a spatial light modulator or a deformable mirror device.
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