Automatic three-dimensional tracking of particles with high-numerical-aperture digital lensless holographic microscopy

We present an automatic procedure for 3D tracking of micrometer-sized particles with high-NA digital lensless holographic microscopy. The method uses a two-feature approach to search for the best focal planes and to distinguish particles from artifacts or other elements on the reconstructed stream of the holograms. A set of reconstructed images is axially projected onto a single image. From the projected image, the centers of mass of all the reconstructed elements are identified. Starting from the centers of mass, the morphology of the profile of the maximum intensity along the reconstruction direction allows for the distinguishing of particles from others elements. The method is tested with modeled holograms and applied to automatically track micrometer-sized bubbles in a sample of 4 mm3 of soda.     

An experimental investigation of three-dimensional particle aggregation using digital holographic microscopy

The tendency of particles to aggregate depends on particle-particle and particle-fluid interactions. These interactions can be characterized but it requires accurate 3D measurements of particle distributions. We introduce the application of an off-axis digital holographic microscopy for measuring distributions of dense micrometer (2 μm) particles in a liquid solution. We demonstrate that digital holographic microscopy is capable of recording the instantaneous 3D position of particles in a flow volume. A new reconstruction method that aids identification of particle images was used in this work. About 62% of the expected number of particles within the interrogated flow volume was detected. Based on the 3D position of individual particles, the tendency of particle to aggregate is investigated. Results show that relatively few particles (around 5–10 of a cohort of 1500) were aggregates. This number did not change significantly with time.     

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.     

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。     

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。     

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.     

High-numerical-aperture microlens shape measurement with digital holographic microscopy

In this Letter, we introduce an algorithm that overcomes limitations in shape measurement by holographic microscopic methods in cases of micro-optical elements with high NA, such as microlenses. The presented algorithm provides a simple method for shape reconstruction from interferometrically measured phase. The algorithm is based on the analysis of local ray transition through the measured object. We develop algorithms for holographic configurations working in transmission and reflection. The accuracy of the developed algorithms is proved by experiments and extensive simulations. We present an experiment in a holographic Mach-Zehnder configuration where we have measured and successfully reconstructed the height distribution of spherical and cylindrical microlenses with NA up to 0.3.     

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.     

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.     

Resolution enhancement in digital holographic microscopy with structured illumination

We present a digital holographic microscope wherein the sample is illuminated by structured light to enable the capture of additional object spatial frequencies. Reconstructed images with increased spatial resolution are obtained by separating and synthesizing bandwidths of different frequency regions in the Fourier domain. The theoretical analysis and experimental results are presented.     

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.     

High-accuracy three-dimensional position measurement of tens of micrometers size transparent microspheres using digital in-line holographic microscopy

Digital in-line holographic microscopy has a strong potential in measuring various three-dimensional (3D) microscale flow phenomena. However, the axial elongation problem in reconstructing particles severely degrades the measurement accuracy along the light propagation direction. In this Letter, we utilize the lenslike characteristic of tens of micrometers size transparent spherical particles to extract their 3D position. A sharp intensity peak is observed in the reconstructed wave field, resulting from the light-focusing effect of the particle. As a result, the depth-of-focus constraint caused by the particle size is eliminated and the measurement accuracy is drastically improved up to submicrometer resolution.     

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.     

Methods of Fourier optics in digital holographic microscopy

In this paper, processing methods of Fourier optics implemented in a digital holographic microscopy system are presented. The proposed methodology is based on the possibility of the digital holography in carrying out the whole reconstruction of the recorded wave front and consequently, the determination of the phase and intensity distribution in any arbitrary plane located between the object and the recording plane. In this way, in digital holographic microscopy the field produced by the objective lens can be reconstructed along its propagation, allowing the reconstruction of the back focal plane of the lens, so that the complex amplitudes of the Fraunhofer diffraction, or equivalently the Fourier transform, of the light distribution across the object can be known. The manipulation of Fourier transform plane makes possible the design of digital methods of optical processing and image analysis. The proposed method has a great practical utility and represents a powerful tool in image analysis and data processing. The theoretical aspects of the method are presented, and its validity has been demonstrated using computer generated holograms and images simulations of microscopic objects.     

An approach for sensing marine plankton using digital holographic imaging

The goal of this paper is to use digital holographic imaging for sensing marine plankton in recording sampling volume. The process stage of this approach includes: wavefront recording using in-line holographic recording set up and numerical reconstruction using Fresnel approximation and convolution algorithm. So, by capturing hologram of marine plankton and reconstructing hologram, the recorded optical field of marine plankton is retrieved. Digital holographic imaging is an extremely powerful technique for the study of marine plankton fields as it allows instantaneous, noninvasive, high-resolution recording of substantial volumes. Finally, this paper presents that it is possible for digital holographic imaging system to sense marine plankton according to laboratory results.     

4-D imaging of fluid flow with digital in-line holographic microscopy

The large depth of field of digital in-line holographic microscopy (DIHM) with numerical reconstruction provides an ideal tool for the study of microfluidic phenomena. As indicators of the flow patterns we use latex microspheres and also red blood cells whose three-dimensional trajectories and velocities can easily be measured as a function of time with subsecond and micron resolution. We demonstrate the efficiency of DIHM by showing 3-D views of the flow patterns around big spheres in various geometric arrangements.     

Extended focused imaging of a microparticle field with digital holographic microscopy

We present a numerical technique for extended focused imaging and three-dimensional analysis of a microparticle field observed in a digital holographic microscope working in transmission. The three-dimensional localization of objects is performed using the local focus plane determination method based on the integrated amplitude modulus. We apply the refocusing criterion locally for each pixel, using small overlapping windows, to obtain the depth map and a synthetic image in which all objects are refocused independent from their refocusing distance. A successful application of this technique in the analysis of the microgravity particle flow experiment is presented.     

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...     

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.