Autofocusing in digital holographic phase contrast microscopy on pure phase objects for live cell imaging

Digital holography enables a multifocus quantitative phase microscopy for the investigation of reflective surfaces and for marker-free live cell imaging. For digital holographic long-term investigations of living cells an automated (subsequent) robust and reliable numerical focus adjustment is of particular importance. Four numerical methods for the determination of the optimal focus position in the numerical reconstruction and propagation of the complex object waves of pure phase objects are characterized, compared, and adapted to the requirements of digital holographic microscopy. Results from investigations of an engineered surface and human pancreas tumor cells demonstrate the applicability of Fourier-weighting- and gradient-operator-based methods for robust and reliable automated subsequent numerical digital holographic focusing.     

Single-shot full resolution region-of-interest (ROI) reconstruction in image plane digital holographic microscopy

We describe a numerical processing technique that allows single-shot region-of-interest (ROI) reconstruction in image plane digital holographic microscopy with full pixel resolution. The ROI reconstruction is modelled as an optimization problem where the cost function to be minimized consists of an L2-norm squared data fitting term and a modified Huber penalty term that are minimized alternately in an adaptive fashion. The technique can provide full pixel resolution complex-valued images of the selected ROI which is not possible to achieve with the commonly used Fourier transform method. The technique can facilitate holographic reconstruction of individual cells of interest from a large field-of-view digital holographic microscopy data. The complementary phase information in addition to the usual absorption information already available in the form of bright field microscopy can make the methodology attractive to the biomedical user community.     

Three-dimensional identification of stem cells by computational holographic imaging.

We present an optical imaging system and mathematical algorithms for three-dimensional sensing and identification of stem cells. Data acquisition of stem cells is based on holographic microscopy in the Fresnel domain by illuminating the cells with a laser. In this technique, the holograms of stem cells are optically recorded with an image sensor array interfaced with a computer and three-dimensional images of the stem cells are reconstructed from the Gabor-filtered digital holograms. The Gabor wavelet transformation for feature extraction of the digital hologram is performed to improve the process of identification. The inverse Fresnel transformation of the Gabor-filtered digital hologram is performed to reconstruct the multi-scale three-dimensional images of the stem cells at different depths along the longitudinal direction. For recognition and classification of stem cells, a statistical approach using an empirical cumulative density function is introduced. The experiments indicate that the proposed system can be potentially useful for recognizing and classifying stem cells. To the best of our knowledge, this is the first report on using three-dimensional holographic microscopy for automated identification of stem cells.     

Microlens characterization by digital holographic microscopy with physical spherical phase compensation

Microlenses have been characterized by a digital holographic microscopy system, which is immune to the inherent wavefront aberration. The digital holographic microscopy system takes advantage of fiber optics and uses the light emitted directly from a single-mode fiber as the recording reference wave. By using such a reference beam, which is quasi-identical to the object beam, the inherent wavefront aberration of the digital holographic microscope is removed. The alignment of the optical setup can be optimized with the help of numerical reconstruction software to give the system phase with the off-axis tilt removed. There is one, and only one, reference fiber point position to give a reference wavefront that is quasi-identical to the object wavefront where the system is free of wavefront aberration and directly gives the quantitative phase of the test object without the need for complicated numerical compensation.     

Determination of the refractive index of dehydrated cells by means of digital holographic microscopy

Spatial distributions of the integral refractive index in dehydrated cells of human oral cavity epithelium are obtained by means of digital holographic microscopy, and mean refractive index of the cells is determined. The statistical analysis of the data obtained is carried out, and absolute errors of the method are estimated for different experimental conditions.     

Parameter-optimized digital holographic microscope for high-resolution living-cell analysis

A parameter-optimized off-axis setup for digital holographic microscopy is presented for simultaneous, high-resolution, full-field quantitative amplitude and quantitative phase-contrast microscopy and the detection of changes in optical path length in transparent objects, such as undyed living cells. Numerical reconstruction with the described nondiffractive reconstruction method, which suppresses the zero order and the twin image, requires a mathematical model of the phase-difference distribution between the object wave and the reference wave in the hologram plane. Therefore an automated algorithm is explained that determines the parameters of the mathematical model by carrying out the discrete Fresnel transform. Furthermore the relationship between the axial position of the object and the reconstruction distance, which is required for optimization of the lateral resolution of the holographic images, is derived. The lateral and the axial resolutions of the system are discussed and quantified by application to technical objects and to living cells.     

Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.     

Digital holographic microtomography with few angle data-sets

In this paper, we report the experimental implementation of handling the reconstruction problem from few angle data-sets for digital holographic microtomography. First, the digital holographic microscopy with sample-rotating scheme is established and few holograms with regularly spaced angle steps are recorded. Then, an algebraic iterative reconstruction algorithm with non-positivity constraint and a smoothing operator is applied to reconstruct three-dimensional refractive index distribution of the measured sample from the few angle data-sets. The experimental results demonstrate that the algebraic iterative technique can accurately reconstruct refractive index distribution from few angle data-sets in digital holographic microtomography. The technique is easy to implement and reduces greatly the required recording times.     

Digital holographic microscope for measuring three-dimensional particle distributions and motions

Better understanding of particle-particle and particle-fluid interactions requires accurate 3D measurements of particle distributions and motions. We introduce the application of in-line digital holographic microscopy as a viable tool for measuring distributions of dense micrometer (3.2 microm) and submicrometer (0.75 microm) particles in a liquid solution with large depths of 1-10 mm. By recording a magnified hologram, we obtain a depth of field of approximately 1000 times the object diameter and a reduced depth of focus of approximately 10 particle diameters, both representing substantial improvements compared to a conventional microscope and in-line holography. Quantitative information on depth of field, depth of focus, and axial resolution is provided. We demonstrate that digital holographic microscopy can resolve the locations of several thousand particles and can measure their motions and trajectories using cinematographic holography. A sample trajectory and detailed morphological information of a free-swimming copepod nauplius are presented.     

Recognition and classification of red blood cells using digital holographic microscopy and data clustering with discriminant analysis

We propose to apply statistical clustering algorithms on a three-dimensional profile of red blood cells (RBCs) obtained through digital holographic microscopy (DHM). We show that two classes of RBCs stored for 14 and 38 days can be effectively classified. Two-dimensional intensity images of these cells are virtually the same. DHM allows for measurement of the RBCs' biconcave profile, resulting in a discriminative dataset. Two statistical clustering algorithms are compared. A model-based clustering approach classifies the pixels of an RBC and recognizes the RBC as either new or old based. The K-means algorithm is applied to the four-dimensional feature vector extracted from the RBC profile.     

Three‐Dimensional Exploration and Mechano‐Biophysical Analysis of the Inner Structure of Living Cells

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self‐interference digital holographic microscopy. This biophotonic holographic workstation enables non‐contact, minimally invasive, flexible, high‐precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 μm diameter are three‐dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.     

Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy

It is shown that the spatial frequencies recorded in interferometric synthetic aperture microscopy do not correspond to exact backscattering [as they do in unistatic synthetic aperture radar (SAR)] and that the reconstruction process based on SAR is therefore based on an approximation. The spatial frequency response is developed based on the three-dimensional coherent transfer function approach and compared with that in optical coherence tomography and digital holographic microscopy.     

Harmonic holography: a new holographic principle

The process of second harmonic generation (SHG) has a unique property of forming a sharp optical contrast between noncentrosymmetric crystalline materials and other types of material, which is a highly valuable asset for contrast microscopy. The coherent signal obtained through SHG also allows for the recording of holograms at high spatial and temporal resolution, enabling whole-field four-dimensional microscopy for highly dynamic microsystems and nanosystems. Here we describe a new holographic principle, harmonic holography (H(2)), which records holograms between independently generated second harmonic signals and reference. We experimentally demonstrate this technique with digital holographic recording of second harmonic signals upconverted from an ensemble of second harmonic generating nanocrystal clusters under femtosecond laser excitation. Our results show that harmonic holography is uniquely suited for ultrafast four-dimensional contrast microscopy.     

Scanning holographic microscopy with resolution exceeding the Rayleigh limit of the objective by superposition of off-axis holograms

We present what we believe to be a new application of scanning holographic microscopy to superresolution. Spatial resolution exceeding the Rayleigh limit of the objective is obtained by digital coherent addition of the reconstructions of several off-axis Fresnel holograms. Superresolution by holographic superposition and synthetic aperture has a long history, which is briefly reviewed. The method is demonstrated experimentally by combining three off-axis holograms of fluorescent beads showing a transverse resolution gain of nearly a factor of 2.     

Polarization microscopy by use of digital holography: application to optical-fiber birefringence measurements

We present a digital holographic microscope that permits one to image polarization state. This technique results from the coupling of digital holographic microscopy and polarization digital holography. The interference between two orthogonally polarized reference waves and the wave transmitted by a microscopic sample, magnified by a microscope objective, is recorded on a CCD camera. The off-axis geometry permits one to reconstruct separately from this single hologram two wavefronts that are used to image the object-wave Jones vector. We applied this technique to image the birefringence of a bent fiber. To evaluate the precision of the phase-difference measurement, the birefringence induced by internal stress in an optical fiber is measured and compared to the birefringence profile captured by a standard method, which had been developed to obtain high-resolution birefringence profiles of optical fibers.     

Resolution improvement by single-exposure superresolved interferometric microscopy with a monochrome sensor

Single-exposure superresolved interferometric microscopy (SESRIM) by RGB multiplexing has recently been proposed as a way to achieve one-dimensional superresolved imaging in digital holographic microscopy by a single-color CCD snapshot [Opt. Lett. 36, 885 (2011)]. Here we provide the mathematical basis for the operating principle of SESRIM, while we also present a different experimental configuration where the color CCD camera is replaced by a monochrome (B&W) CCD camera. To maintain the single-exposure working principle, the object field of view (FOV) is restricted and the holographic recording is based on image-plane wavelength-dispersion spatial multiplexing to separately record the three bandpass images. Moreover, a two-dimensional extension is presented by considering two options: time multiplexing and selective angular multiplexing. And as an additional implementation, the FOV restriction is eliminated by varying the angle between the three reference beams in the interferometric recording. Experimental results are reported for all of the above-mentioned cases.     

Transverse resolution improvement using rotating-grating time-multiplexing approach

The ability to improve the limited resolving power of optical imaging systems while approaching the theoretical diffraction limit has been an attractive discipline with growing interest over the last years due to its benefits in many applied optics systems. This paper presents a new approach to achieve transverse superresolution in far-field imaging systems, with direct application in both digital microscopy and digital holographic microscopy. Theoretical analysis and computer simulations show the validity of the presented approach.     

Holographic digital Fourier microscopy for selective imaging of biological tissue

We present an application of digital Fourier holography for selective imaging of scatterers with different sizes in turbid media such as biological tissues. A combination of Fourier holography and high‐resolution digital recording, digital Fourier microscopy (DFM) permits crucial flexibility in applying filtering to highlight scatterers of interest in the tissue. The high‐resolution digital hologram is a result of the collation of Fourier holographic frames to form a large‐size composite hologram. It is expected that DFM has an improved signal‐to‐noise ratio as compared to conventional direct digital imaging, e.g., phase microscopy, as applied to imaging of small‐size objects. The demonstration of the Fourier filtering capacity of DFM using a biological phantom represents the main focus of this article. © 2005 Wiley Periodicals, Inc. Int J Imaging Syst Technol, 14, 253–258, 2004; Published online inWiley InterScience (www.interscience.wiley.com). DOI 10.1002/ima.20031     

Magnified reconstruction of digitally recorded holograms by Fresnel-Bluestein transform

A method for numerical reconstruction of digitally recorded holograms with variable magnification is presented. The proposed strategy allows for smaller, equal, or larger magnification than that achieved with Fresnel transform by introducing the Bluestein substitution into the Fresnel kernel. The magnification is obtained independent of distance, wavelength, and number of pixels, which enables the method to be applied in color digital holography and metrological applications. The approach is supported by experimental and simulation results in digital holography of objects of comparable dimensions with the recording device and in the reconstruction of holograms from digital in-line holographic microscopy.