Abstract
Scattering and absorption belong to the major problems in imaging the internal layers of a biological specimen. Due to the structural inhomogeneity of the specimen, the distribution of the structures in the upper layers of a given internal structure of interest is different from the lower layers that may result in different interception of scattered light, falling into the angular aperture of the microscope objective, from the object in each imaging view. Therefore, different spatial frequencies of the scattered light can be acquired from different (top and bottom) views. We have arranged an opposed-view dark-field digital holographic microscope (DHM) to collect the scattered light concurrently from both views with the aim to increase the contrast of internal structures and improve the signal-to-noise ratio. Implementing a DHM system gives the possibility to implement digital refocusing process and obtain multilayer images from each side without a depth scan of the object. The method is explained and the results are presented exemplary for a Drosophila embryo.
Highlights
Imaging internal layers of biological specimens is a demanding field of research [1]
Scattering and absorption belong to the major problems in imaging the internal layers of a biological specimen
Due to the structural inhomogeneity of the specimen, the distribution of the structures in the upper layers of a given internal structure of interest is different from the lower layers that may result in different interception of scattered light, falling into the angular aperture of the microscope objective, from the object in each imaging view
Summary
Imaging internal layers of biological specimens is a demanding field of research [1]. Scan-free methods like digital holographic microscopy (DHM) have been introduced to extract the object wave-front leading to a multilayer image of a specimen via digital refocusing and quantitative phase (topographic) image [3, 4]. One of the major issues in imaging the internal structures of biological specimens is the absorption and scattering that distort the information coming from the object. Some techniques have been developed to reduce this effect, based on non-linear light-matter interaction like two-photon microscopy [8], or confining the excitation area to a selective internal layer using light sheet microscopy [9, 10]
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