基于正交双波长测量的有核细胞三维形态重建

Three-Dimensional Morphological Reconstruction of Nucleated Cell Based on Orthogonal Dual-Wavelength Measurement

细胞是有机体结构与生命活动的基本单位,其形态结构往往与功能状态相匹配,因而细胞的形态是生命科学的重要研究内容,对临床医学诊断也有着重要的意义。定量相位成像技术作为一种强大的无损、无标记成像工具,可为定量评估细胞提供重要的生物、物理特性信息。提供的相位图虽携带了样品内部结构信息,但样品厚度与折射率耦合在相位数据中,需要借助算法对相位数据进行解耦,才能重建样品的三维形态。提出了基于两路正交的双波长相位信息重建多介质相位体三维形态的方法。以有核生物细胞为例,利用该方法对样本的两幅同方向双波长相位图按亚结构分布进行区域划分,对应解析不同区域内参考点的相位值,提取出胞质折射率及其沿入射光方向的厚度分布,据此分离胞质和胞核各自引起的相位;进一步联合正交方向的相位图,获取胞核中参考点在对应方向上的物理厚度,并相继计算出胞核的平均折射率和胞核整体厚度分布,从而利用两个正交方向的三幅相位图重建出样品的三维形态。

Objective Cells are the basic units of biomass,and their morphological structures are often associated with the functional state of biomass.Therefore,the morphology of a cell is an important research topic in life science and a critical factor in clinical medical diagnosis.Quantitative phase imaging technology,as a powerful nondestructive and label-free imaging tool,provides various biological and physical properties for the quantitative evaluation of cells.Although the phase diagram of the sample provided by this technology contains information about its internal structure,the thickness and refractive index of the sample is coupled with the phase data.Decoupling the phase data is required to reconstruct a three-dimensional(3D)morphology of the sample.Dual-wavelength imaging technology is effective for single medium samples.However,this method does not work for multimedia phase objects.In response to this shortage,this study proposes a new reconstruction method based on orthogonal dual-wavelength measurements.Methods The 3Dreconstruction method is based on three phase images from two orthogonal directions.Two of these phase images are obtained with two different wavelengths.The first step is to separate the phase shift due to different substructures.Given that the environmental liquid is a highly dispersive material relative to the cell sample,the refractive index(RI)of the environmental liquid correspondingly changes under different incident light,whereas the RI of the sample remains constant.Thus,by subtracting the two images at two different wavelengths,the physical thickness of the media adjacent to the environment(such as cytoplasm)can be determined.Next,the average RI of the cytoplasm can be extracted using the associated phase value distribution,while phase shifts due to cytoplasm and nucleus are also separated immediately.Following that,the thickness information of the nucleus for the incidence along the two directions can be obtained using aphase diagram from the orthogonal direction.Thus,the RI of the nucleus can be calculated from the nuclear phase value.The relative position of the cytoplasm and nucleus can also be determined using two orthogonal phase diagrams.The 3D morphology of the multimedia phase object is obtained by combining the physical thickness distributions of the cytoplasm and nucleus.Results and Discussions The reconstructions of models with different structural characteristics are explored,including a cell with a single spherical nucleus(Fig.2),a cell with a single saddle shape nucleus[Fig.5(a)],and a binuclear cell with a double spherical nucleus[Fig.6(a)].The results of these samples[Figs.9,11(c),and 12(e)]are consistent with the initial model.Especially,the analytic method provides a sharp reconstruction result of the physical thickness of the cytoplasm and the entire reconstruction process takes a short time(Tables 1,2,and 3).This study suggests the feasibility of this reconstruction method,but the actual application effect depends on many factors,such as image noise,heterogeneity of RI distribution,and calculation error in edge detection.An emphasis of the following study is to explore an efficient reconstruction algorithm suitable for experiments.Conclusions This study proposes a 3D morphological reconstruction method for nucleated cells based on orthogonal dual-wavelength phase images.This method requires three phase images from two orthogonal directions and is divided into two steps.First,using the high dispersion characteristics of environmental liquid and edge detection,the phases of the cytoplasm and nucleus are separated based on the independence and superposition of phase data,and the thickness of the cytoplasm is decoupled simultaneously.Then,the 3D morphology of the sample is reconstructed using another orthogonal phase diagram,RI and thickness information of the coupling nucleus,and the relative position relationship of the substructure expressed by two mutually orthogonal phase diagrams.This method collects sample information from two directions simultaneously.A small amount of data means convenient data acquisition and fast data processing.The simulation results show that the algorithm,which may provide a reference for real-time imaging of biological cells,is effective.