Abstract
Unlike mammalian erythrocytes, amphibian erythrocytes have distinct morphological features including large cell sizes and the presence of nuclei. The sizes of the cytoplasm and nuclei of erythrocytes vary significantly over different species, their environments, or pathophysiology, which makes hematological studies important for investigating amphibian species. Here, we present a label-free three-dimensional optical quantification of individual amphibian erythrocytes from frogs Pelophylax nigromaculatus (Rana nigromaculata). Using optical diffraction tomography, we measured three-dimensional refractive index (RI) tomograms of the cells, which clearly distinguished the cytoplasm and nuclei of the erythrocytes. From the measured RI tomograms, we extracted the relevant biochemical parameters of the cells, including hemoglobin contents and hemoglobin concentrations. Furthermore, we measured dynamic membrane fluctuations and investigated the mechanical properties of the cell membrane. From the statistical and correlative analysis of these retrieved parameters, we investigated interspecific differences between frogs and previously studied mammals.
Highlights
The properties of erythrocytes are related to the cellular functions in the circulatory system
Similar to X-ray computed tomography (CT), optical diffraction tomography (ODT) reconstructs the 3D tomogram of a sample from multiple 2D images of the sample obtained with various illumination angles[28]
Based on the Fourier diffraction theorem with Rytov approximation[45], the 3D refractive index (RI) tomogram of the sample is reconstructed from the retrieved amplitude and phase images (Fig. 1d)
Summary
The properties of erythrocytes are related to the cellular functions in the circulatory system. In the evolutionary point of view, hematological studies of amphibians play an important role in the study of their phylogenetic pathways following the vertebrate invasion of the land[1] One of their critical traits of amphibians, compared to other vertebrates, is their unusually large erythrocytes, which may be associated with the adaptation for the transition from aquatic to terrestrial life. Bright-field microscopy has been used to quantify the size parameters of the cells in two-dimensions (2D), such as their aspect ratios[5] This method has limitations when assessing the three-dimensional (3D) information of a cell. Quantitative phase imaging (QPI) techniques have been developed as a label-free imaging method for the study of live cells and tissues[8,9]. One particular example is the amphibian erythrocyte, whose morphological and chemical properties of the nuclei and the cytoplasm have not been investigated using 3D QPI
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