Abstract
We show how a pyramid phase microscope can be used to obtain tomographic information of the spatial variation of refractive index in biological samples using the Radon transform. A method that uses the information provided by the phase microscope for axial and lateral repositioning of the sample when it rotates is also described. Its application to the reconstruction of mouse embryos in the blastocyst stage is demonstrated.
Highlights
In recent years, numerous results have appeared describing the tomographic reconstruction of fluorescent markers in small biological samples or whole organisms, offering invaluable information on gene expression localization and the spatial distributions of molecules
We show how a pyramid phase microscope can be used to obtain tomographic information of the spatial variation of refractive index in biological samples using the Radon transform
A method that uses the information provided by the phase microscope for axial and lateral repositioning of the sample when it rotates is described
Summary
Numerous results have appeared describing the tomographic reconstruction of fluorescent markers in small biological samples or whole organisms, offering invaluable information on gene expression localization and the spatial distributions of molecules. Independently of the method used, transillumination phase maps represent – neglecting diffraction effects– the integrated phase, i.e., the sum of the different retardations that the tissue induces in the light path, not the three-dimensional distribution of optical density This problem has been considered before and reconstruction algorithms have been developed and applied to the phase data obtained with a variety of techniques: interferograms registered for different illumination angles [9,10,11], lens-free holograms acquired from a multiplicity of angles [12], diffraction images obtained by focus scanning [13], training a neural network to infer the three-dimensional index distribution [14] or rotating the object, estimating the phase by the transport of intensity equation [15].
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