Measurement of refractive index change induced by dark reaction of photopolymer with digital holographic quantitative phase microscopy

Light-induced refractive index change in photopolymer is quantified by a digital holographic microscope. The refractive index change is induced as the dark reaction which proceeds inside the photopolymer after a writing beam is stopped. Time-lapse phase distribution across the photopolymer is measured by the off-axis digital holographic microscope which enables us to retrieve the 2-D phase map from a single hologram. It is found that the initial phase profile does not coincide with the illumination intensity distribution. This observation suggests that the rate of the refractive index change in dark reaction is not proportional to the illumination intensity in case the exposure energy becomes high.     

Laser-ablation-induced refractive index fields studied using pulsed digital holographic interferometry

Pulsed digital holographic interferometry has been used to investigate the plume and the shock wave generated in the ablation process of a Q-switched Nd-YAG (λ=1064 nm and pulse duration=12 ns) laser pulse on a polycrystalline boron nitride (PCBN) target under atmospheric air pressure. A special setup based on two synchronised wavelengths from the same laser for simultaneous processing and measurement has been used. Digital holograms were recorded for different time delays using collimated laser light (λ=532 nm) passed through the volume along the target. Numerical data of the integrated refractive index field were calculated and presented as phase maps showing the propagation of the shock wave and the plume generated by the process. Radon inversion has been used to estimate the 3D refractive index fields measured from the projections assuming rotational symmetry. The shock wave density has been calculated using the point explosion model and the shock wave condition equation and its behaviour with time at different power densities ranging from 1.4 to 9.1 GW/cm² is presented. Shock front densities have been calculated from the reconstructed refractive index fields using the Gladstone–Dale equation. A comparison of the shock front density calculated from the reconstructed data and that calculated using the point explosion model at different time delays has been done. The comparison shows quite good agreement between the model and the experimental data. Finally the reconstructed refractive index field has been used to estimate the electron number density distribution within the laser-induced plasma. The electron number density behaviour with distance from the target at different power densities and its behaviour with time are shown. The electron number densities are found to be in the order of 10¹⁸ cm^?3 and decay at a rate of 3×10¹⁵ electrons/cm³ ns.     

Simultaneous cell morphometry and refractive index measurement with dual-wavelength digital holographic microscopy and dye-enhanced dispersion of perfusion medium

Digital holographic microscopy (DHM) allows optical-path-difference (OPD) measurements with nanometric accuracy. OPD induced by transparent cells depends on both the refractive index (RI) of cells and their morphology. This Letter presents a dual-wavelength DHM that allows us to separately measure both the RI and the cellular thickness by exploiting an enhanced dispersion of the perfusion medium achieved by the utilization of an extracellular dye. The two wavelengths are chosen in the vicinity of the absorption peak of the dye, where the absorption is accompanied by a significant variation of the RI as a function of the wavelength.     

Immersion digital in-line holographic microscopy

Digital in-line holographic microscopy is a promising new tool for high resolution imaging. We demonstrate, by using latex beads, that a considerable increase in numerical aperture, and, therefore, resolution can be achieved if the space between a source and a CCD camera chip is filled with a high refractive index medium. The high refractive index medium implies a shorter effective wavelength so that submicrometer resolution can be obtained with laser light in the visible range.     

Multiframe full-field heterodyne digital holographic microscopy

Digital holographic microscopy using multiframe full-field heterodyne technology is discussed in which two acousto-optic modulators are applied to generate low-frequency heterodyne interference and a high-speed camera is applied to acquire multiframe full-field holograms. We use a temporal frequency spectrum analysis algorithm to extract the object's information. The twin-image problem can be solved and the random noise can be significantly suppressed. The relationship between the frame number and the reconstruction accuracy is discussed. The typical objects of microlenses and biology cells are reconstructed well with 100-frame holograms for illustration.     

Dual plane in-line digital holographic microscopy

We report a dual plane in-line digital holographic microscopy technique that exploits the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. Two interferograms are recorded at different planes to eliminate the conjugate image. The experimental results demonstrate successful reconstruction of phase objects as well as of amplitude objects. The two interferograms can be recorded simultaneously, using two CCD or CMOS sensors, in order to increase the acquisition rate. This enhanced acquisition rate, together with the improved reconstruction capability of the proposed method, may find applications in biomedical research for visualization of rapid dynamic processes at the cellular level.     

基于涡旋光照明的暗场数字全息显微方法研究

提出了一种基于涡旋光照明的暗场数字全息显微方法. 从理论上阐述了涡旋光环形照明原理和暗场数字全息显微原理, 分析了涡旋光的准无衍射特性对成像的影响; 搭建了相应的数字全息显微成像系统, 采用690 nm的聚苯乙烯小球作为实验样品; 最后通过对小球明暗场下数字全息显微再现像的分析对比, 证明该方法可以有效地提高数字全息系统的分辨率, 同时增强了再现像的对比度. 关键词： 全息 暗场数字全息显微 涡旋光 分辨率     

Polarization phase shifting in digital holographic microscopy

In the present work we have made use of polarization phase shifting in digital holographic microscopy (DHM) for three dimensional phase profiling of transmissive and reflecting microscopic samples. The Mach–Zehnder arrangement with proper polarizing elements (polarizer-masked cube beam splitter, quarter wave plate and a linear polarizer) is used for recording the phase-shifted digital holograms. The suggested procedure is simple and accurate and obviates the need of piezo devices for phase shifting. The phase profile of the specimen is reconstructed from the holograms by using standard phase shifting algorithms.     

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 by spectrum centering in digital holographic microscopy

Firstly, an approach is proposed for eliminating the tilt phase aberration introduced by the tilt reference wave. It is based on the argument distribution of the hologram spectrum to locate exactly the position of the carrier frequency of the virtual image. This tilt aberration will be corrected by shifting the filtered hologram spectrum to the coordinate origin of the frequency domain, without knowledge of the focal length of the imaging lens or distances in the setup. Then the subsequent quadratic phase aberration compensation is performed only by adjusting a single parameter. The method is demonstrated by the phase contrast imaging of the cervical carcinoma cells.     

Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy

Optical Review - Hybrid digital holographic microscopy that combines fluorescence microscopy and digital holographic microscopy into a single system for biological applications is proposed. In the...     

少量投影数字全息层析重建实验研究

提出了少量投影数字全息层析重建技术.以具有轴对称结构的光纤和非轴对称结构的石膏头像为实验样本,开展了基于单幅全息图和代数迭代重建算法的层析重建模拟分析及实验研究.模拟分析表明,代数迭代重建算法中的加权因子以及松弛因子对重建图像质量影响显著,因此对加权因子采用非线性加权计算方法,以及通过多次模拟重建结果的比较选择合适的松弛因子.层析重建实验结果表明,无论被测物体是轴对称结构或是非轴对称结构,少量投影数字全息层析重建是可行的,也是有效的.这为下一步针对生物样本内部多层折射率检测和基于单幅层析全息图实现三向实时数字全息层析重建技术研究提供了必要的基础.     

Complex-amplitude-based phase unwrapping method for digital holographic microscopy

A complex-amplitude-based phase unwrapping approach for digital holographic microscopy is proposed in this paper. A quality map is derived directly from the reconstructed complex amplitude distribution of object wave to evaluate noise influence and phase reliability in the wrapped phase image. Quality-guided phase unwrapping algorithm is then implemented with the quality map to retrieve continuous phase profile. Unwrapping errors caused by unreliable phase data are successfully suppressed. The effectiveness of this method is demonstrated with simulation and experimental results.     

Disturbance-free digital holographic microscopy via a micro-phase-step approach

This work proposes a cost-effective, simple, micro-phase-step (MPS) method for suppressing the zero-order diffraction and conjugate-image interferences that are caused during digital holographic microscopic image reconstruction. The proposed MPS method replaces the conventional phase modulation approach; it uses a rotatable cover glass that enables smooth modification of the incidence angle and the optical path of the reference beam. This setup allows the phase step to be accurately estimated by shifting the reference wave phase more freely close to π/2, at which the background noise can be suppressed more effectively. In the proposed MPS method, the optimal conditions for suppressing conjugate-image interference are identified using a relatively moderate intensity distribution and suppression of noise in the numerically reconstructed object wave-field. In addition, the proposed method mitigates the effect of disturbances that are caused by environmental factors, such as minor vibrations and small changes in temperature and humidity. Importantly, only two holograms are required to satisfy the objective of image reconstruction. The results in this work reveal that even with intentional interference caused by minor vibrations, conjugate-image interference can still be suppressed by determining the phase deviation between the two original holograms.     

Long-term quantitative phase-contrast imaging of living cells by digital holographic microscopy

The dynamic analysis of biological living samples is one of the particular interests in life sciences. An improved digital holographic microscope for long-term quantitative phase-contrast imaging of living cells is presented in this paper. The optical configuration is optimized in the form of a free-space-fiber hybrid system which promotes the flexibility of imaging in complex or semi-enclosed experimental environment. Aberrations compensation is implemented taking into account the additional phase aberration induced by liquid culture medium in long-term observation. The proposed approach is applied to investigate living samples of MC3T3-E1 and MLO-Y4 cells. The experimental results demonstrate its availability in the analysis of cellular changes.     

预放大数字全息显微系统的特点

为了提高预放大数字全息显微系统的成像质量,采用理论分析与实验验证相结合的方法,分别对采用平面及球面参考光记录的预放大数字全息显微系统进行了研究和比较。结果表明：在通常的实验条件下,系统的横向分辨力主要取决于显微物镜的成像分辨力;记录距离较小时,两种系统的横向分辨力均随着记录距离的增大略有降低;但当记录距离较大时,球面参考光预放大数字全息系统的横向分辨力降低得更为明显,即平面参考光预放大数字全息显微系统较为优越;在记录距离为0的情况下,即像面数字全息成像情况下,两种系统的再现像均具有最高的分辨力,在利用普通工业用传感器条件下,横向分辨力远超过了2.19 m,且像质较好。因此,尽可能减小全息图的记录距离,或者采用像面数字全息系统,可以有效提高数字全息系统的成像分辨力。     

Time-resolved digital holographic microscopy of laser-induced forward transfer

We develop a method for time-resolved digital holographic microscopy to obtain time-resolved 3-D deformation measurements of laser-induced forward transfer (LIFT) processes. We demonstrate nanometer axial resolution and nanosecond temporal resolution of our method which is suitable for measuring dynamic morphological changes in LIFT target materials. Such measurements provide insight into the early dynamics of the LIFT process and a means to examine the effect of laser and material parameters on LIFT process dynamics.     

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.     

Color lensless digital holographic microscopy with micrometer resolution

Color digital lensless holographic microscopy with micrometer resolution is presented. Multiwavelength illumination of a biological sample and a posteriori color composition of the amplitude images individually reconstructed are used to obtain full-color representation of the microscopic specimen. To match the sizes of the reconstructed holograms for each wavelength, a reconstruction algorithm that allows for choosing the pixel size at the reconstruction plane independently of the wavelength and the reconstruction distance is used. The method is illustrated with experimental results.     

Label-free second-harmonic phase imaging of biological specimen by digital holographic microscopy

We have previously developed a new way for nonscanning second-harmonic generation (SHG) microscopy [Opt. Lett. 34, 2450 (2009)]. Based on digital holography, this technique captures, in single-shot hologram acquisition, both the amplitude and the phase of a coherent SHG radiation, which makes possible second harmonic phase microscopy. In this work, we present holographic SHG phase microscopy of a label-free biological tissue and discuss its added value to SHG microscopy.