数字全息显微术对细胞的研究

通过数字全息显微的方法分析研究了新鲜洋葱表皮细胞的形貌结构. 根据全息理论、光的衍射理论和透镜的相位变换性质,从理论上分析了数字全息显微原理,并依据四步相移和最小二乘去包裹技术,研究了重构细胞相位的方法.分析比较了不同参考光对数字全息显微分辨率的影响,设计了用球面光波作为参考光记录数字全息显微的装置.以新鲜洋葱表皮细胞为样本完成了实验检测和相位重构,得到了细胞的相位和三维信息,并粗略估计了细胞宽度和厚度.系统分辨率达到了1.3 μm.     

基于相移技术的显微数字全息重构细胞相位

介绍了用显微镜物镜、压电陶瓷和CCD建立的一套测量细胞相位的显微数字全息光路,基于相移技术,给出了重构相位的理论分析,并用洋葱磷片叶细胞作为测试样品,完成了测量细胞相位的实验.结果表明：该系统可以完成细胞相位重构,系统分辨率不低于1 μm.     

单光路共轴数字全息去背景新方法及应用

针对数字全息图像背景去除的问题,提出两种全息图像重建去背景的方法:背景减弱法和自适应滤波法.搭建了共轴数字全息显微成像实验系统,利用该系统分别对洋葱表皮、植物根茎横切标本、叶片气孔标本和血细胞标本进行背景去除和图像重建.通过光强分布曲线、对比度计算等证明了自适应滤波方法在共轴数字全息结构中去除背景的有效性.实验结果表明,根据环境、样品等不同,全息图的背景也会发生变化.基于此结果,将全息数字显微图像分为三类,并针对不同的背景特点,得出了获取最佳重建图像应采用的有效去除背景方法.研究结果有助于共轴光路数字全息显微术中的重建成像.     

数字全息显微术中重建物场波前的相位校正

数字全息显微术克服了传统光学显微术无法直接提取样品相位信息的缺点,可以对活体细胞组织等相位型生物样品进行定量测量和有效观察。但在数字全息显微成像过程中,像场弯曲会对再现像相位分布的测量和观察产生影响。提出一种采用相位相减来校正数字全息再现像像场弯曲的方法。通过在样品加入前后两次拍摄全息图,并对数值重建像分别进行去包裹运算再令其相减,即可实现对像场弯曲的有效校正,对蝉翼和大蒜表皮细胞等相位型物体进行测量,并采用数值校正和相位相减两种方法对像场弯曲进行校正。与现有的数值校正方法相比,利用相位相减获得样品三维相位信息的方法更为简单、可靠,是校正像数字全息再现场弯曲的有效方法。     

结合划线拟合和深度学习的数字全息显微相位像差自动补偿方法

针对动态观察活体细胞形态的数字全息显微应用,提出了一种结合划线拟合和深度学习的自动相位像差补偿方法.首先在全息面提取中心十字线上的物光场相位值,通过二次多项式数值拟合构建相位掩模完成相位像差初步补偿.然后在成像面运用卷积神经网络生成二值化图像掩膜,提取物光场中无物体区域的相位值,再次通过高阶多项式进行数字拟合构建相位掩模完成相位像差精确补偿.最后得到无相位像差的再现相位像.该方法通过在全息面划线取值和数字拟合有效补偿物光中的主要相位像差,降低了成像面物光场的图像轮廓复杂性,利用有限的训练数据集获得能够准确建立图像分割的深度学习模型,从而实现了准确的无需人工干预的数字全息显微自动相位像差补偿.基于离轴数字全息显微成像系统对多种具有不同形态特征的活体细胞开展动态观察实验,并进一步应用该方法进行子宫内膜癌细胞抗药性筛选.结果表明该方法可以很好地用于动态显微观察,从而为生物学细胞动态研究提供实验依据.     

基于液晶空间光调制器的相移数字全息显微测量系统精度分析

液晶空间光调制器的空间不均匀性和边缘场效应导致调制后相位存在畸变,为提高基于液晶空间光调制器的相移数字全息显微系统的测量精度,提出了二次相位标定相移数字全息的测量方法。首先,基于斐索干涉系统,以反射式液晶空间光调制器为被测对象,对其灰度与相移量的关系进行一次标定,并测量畸变波前相位;然后,利用二维图像插值算法及灰度化算法,设计出与液晶空间光调制器分辨率相等的畸变相位共轭灰度图;最后,将共轭灰度图加载在液晶空间光调制器上,获得理想调制相位,达到二次相位标定的目的。构建基于液晶空间光调制器的相移数字全息显微测量系统,对微透镜阵列进行测量实验,结果表明：二次标定前后微透镜阵列纵向矢高的相对测量误差由3.08%减少到1.15%,证明该方法能够有效提高相移数字全息的测量精度。     

利用综合复用技术拓展数字全息显微系统中的记录视场

为了解决现有数字全息显微系统中高分辨率与大记录视场无法同时兼得的问题, 提出了一种在不牺牲分辨率的前提下拓展数字全息显微记录视场的方法. 该方法中运用了波长不同、偏振态不同的四路相互不相干的探测物光, 同时探测被测样品四个相邻的不同区域, 并使这四束探测物光分别与其相应的参考光相干, 在记录面上同时记录下含有被测样品不同区域信息的复合全息图. 将获得的复合全息图经过频谱变换和数字滤波, 分别重构出所记录区域的振幅和相位分布.最后通过图像拼接和图像融合技术, 可实现接近原记录视场四倍的大视场数字全息显微记录. 该方法在测量过程中无需移动记录装置、光源和被测样品, 单次曝光即可实现, 实验结果验证了本文所提方法的可行性. 关键词： 数字全息显微术 角分复用 偏振复用 波长复用     

数字显微全息中二次项相位误差的补偿

针对预放大式数字离轴显微全息技术中二次项相位误差,提出一种基于单幅全息再现的补偿方法.该方法是在显微物镜后焦平面处实现该相位误差的数学补偿,相位误差补偿过程具有调整参量单一化的优点.同时,利用显微物镜的后焦平面上物点特征实现了最佳再现距离的自动判断.该文以相位光栅为实验样本,对该方法进行了实验例证,实验结果验证了本文所提出二次项相位误差的补位误差的补偿方法的正确性和有效性.     

基于数字显微全息技术的相位光栅结构测量

以相位光栅为实验对象,开展了基于数字显微全息技术的相位物体三维显微结构信息的再现研究.在Mach-Zender透射式实验系统的基础上,分别采用显微物镜和无透镜放大方式,对相位光栅进行放大,以提高系统横向分辨率.在显微物镜放大系统中,菲涅耳近似数值再现算法与双波长技术相结合,抑制主要系统噪音,获得相位光栅的显微结构三维分布.在无透镜放大数字显微全息系统中,分别利用菲涅耳近似法和卷积方法再现原始物波前,并提出相减法消除系统主要球面误差,获得相位光栅的深度信息.实验结果与Veeco干涉仪测试结果比对表明,光栅周期和深度结构与干涉仪测试数据相符.     

利用小尺寸电荷耦合器件实现数字全息高分辨成像

为了在降低数字全息显微成像系统成本的同时实现高分辨成像,对像面数字全息显微术的记录与再现过程进行了理论分析,结合系统的点扩散函数,对该系统的横向分辨率进行了分析. 得到了如下结论：像面数字全息显微系统的横向分辨率对电荷耦合器件（CCD）光敏面尺寸变化不敏感;对于常见的CCD 器件,其像元尺寸的变化对该系统的横向分辨率影响甚微. 此外,对像面数字全息显微系统的成像特点进行了分析,结果表明：利用像面数字全息系统可以实现物体信息的完整记录与再现,其成像分辨率及像质优于预放大数字全息系统. 利用搭建的数字全息实验记录系统,从强度及位相两方面对理论分析结果进行了验证,实验结果表明了理论分析的正确性. 关键词： 像面数字全息术 横向分辨率 点扩散函数 小尺寸电荷耦合器件     

Phase aberration compensation of digital holographic microscopy based on least squares surface fitting

Digital holographic microscopy allows the numerical reconstruction of the complex wavefront of samples, especially biological samples such as living cells. In digital holographic microscopy, a microscope objective is introduced to improve the transverse resolution of the sample; however a phase aberration in the object wavefront is also brought along, which will affect the phase distribution of the reconstructed image. We propose here a numerical method to compensate for the phase aberration of thin transparent objects with a single hologram. The least squares surface fitting with points number less than the matrix of the original hologram is performed on the unwrapped phase distribution to remove the unwanted wavefront curvature. The proposed method is demonstrated with the samples of the cicada wings and epidermal cells of garlic, and the experimental results are consistent with that of the double exposure method.     

Digital self-referencing quantitative phase microscopy by wavefront folding in holographic image reconstruction

A completely numerical method, named digital self-referencing holography, is described to easily accomplish a quantitative phase microscopy for microfluidic devices by a digital holographic microscope. The approach works through an appropriate numerical manipulation of the retrieved complex wavefront. The self-referencing is obtained by folding the retrieved wavefront in the image plane. The folding operation allows us to obtain the correct phase map by subtracting from the complex region of interest a flat area outside the microfluidic channel. To demonstrate the effectiveness of the method, quantitative phase maps of bovine spermatozoa and in vitro cells are retrieved.     

Phase reconstruction in lensless digital in-line holographic microscopy

The phase reconstruction in a digital in-line holographic microscopy is compared using two numerical reconstruction methods. The first method uses one Fourier transform and second one uses three Fourier transforms. It is shown that the latter method gives improved object phase reconstruction as compared to the former.     

Characterisation of light emitting diodes (LEDs) for application in digital holographic microscopy for inspection of micro and nanostructured surfaces

Partial coherent light sources open up prospects for phase noise reduction in digital holographically reconstructed phase distributions by suppressing multiple reflections in the experimental setup. Thus, light emitting diodes (LEDs) are investigated for application in digital holographic microscopy. First, the spectral properties and the resulting coherence length of an LED are characterised. In addition, an analysis of dispersion effects and their influence on the hologram formation is carried out. The coherence length of LEDs in the range of a few micrometers restricts the maximum interference fringe number in off-axis holography for spatial phase shifting. Thus, the application of temporal phase-shifting-based digital holographic reconstruction techniques is compared to spatial phase-shifting-based methods. It is demonstrated that LEDs are applicable for digital holographic microscopy in connection with both spatial and temporal phase-shifting-based techniques for reduction of noise in comparison to a laser-light-based experimental setup.     

Dual-wavelength linear regression phase unwrapping in three-dimensional microscopic images of cancer cells

We present a study of the three-dimensional structure of cancer cells using dual-wavelength phase-imaging digital holographic microscopy. Phase imaging of objects with optical height variation greater than the wavelength of light is ambiguous and causes phase wrapping. By comparing two phase images recorded at different wavelengths, the images can be accurately unwrapped. The unwrapping method is computationally fast and straightforward, and it can process complex topologies. Additionally, the limitations on the total optical height are significantly relaxed. This new methodology is widely applicable to other phase-imaging techniques as well as in applications beyond optical microscopy.     

傅立叶变换用于全息三维物体面形测量

介绍了将傅立叶变换用于全息三维面形测量的基本原理和实验装置。首先用全息干涉条纹替代投影条纹投影到待测物体上 ,在相位解调中 ,结合Gerchberg迭代算法对变形条纹进行处理 ,然后用汉宁窗口进行数字加权滤波。结果表明 ,该测量系统实验装置简单 ,测量精度高 ,速度快 ,适用于复杂大物体三维面形测量     

Application of time-resolved digital holographic microscopy in studies of early femtosecond laser ablation

We studied evolution of femtosecond laser ablation by employing novel method of time-resolved off-axis digital holographic microscopy. Phase and amplitude profiles of early shock front and ablation plume dynamics of irradiated tempered steel were reconstructed from the digital holograms. In order to gain additional information, digital holographic microscopy was combined with plasma emission imaging. By using both techniques simultaneously we studied material response to multi-pulse irradiation, shock wave propagation, ablation plume formation and plasma emission. The significant changes in ablation performance were observed when using multi-pulse irradiation if compared to widely investigated single-shot regime.     

Internal inspection of semi-transparent objects by digital holographic micro-tomography

The combined use of digital holographic microscopy and computer tomography, here named digital holographic micro-tomography, is used to examine the interior of transparent channels. The proposed method is used to identify internal obstacles inside of transparent troughs having slightly different refractive index. The method is based in the acquisition of a set of digital holograms of the specimen whereas it is axially rotated from 0° to 180°. The phase differences retrieved from the obtained holograms are the inputs to a computerised axial tomography procedure. The technique has been numerically modelled in order to find the optimal tomographic conditions and also to realise the minimum difference of refractive index the method could detect. The obtained results show the feasibility of the proposed method for the non-destructive evaluation of transparent micro-objects.