Phase-shifting Digital Holographic Microscopy Research Articles

3D dynamic observation of human sperm by parallel phase-shifting digital holographic microscopy based on pixelated polarization

Morphology and motility are essential criteria for assessing sperm viability. However, the human sperm head is small (∼3–4 μm) and requires a relatively high-magnification microscope objective, while the sperm flagella (∼45 μm) are poorly visible with complex 3D properties. Microscopic dynamic observation of intact sperm in 3D is challenging. Conventional inspection methods with a limited depth of field are inadequate for this issue. To provide a solution to this critical need, we develop pixelated polarization-based parallel phase-shifting digital holographic microscopy for the 3D dynamic observation of human sperm. Compared to conventional holographic imaging, this approach can effectively separate the object wavefront and avoid image quality degradation while fully exploiting the spatial bandwidth of the camera. We propose the use of the Stokes parameter reconstruction method to reconstruct the object wavefront and investigate the effect of the sampling interval on the system resolution by spectral analysis. The methodology achieves the retrieval of the 3D trajectory and motion parameters of sperm and reconstructs the sperm head orientation and the thin, highly-dynamic flagellum. The system allows for more comprehensive information on sperm motility and morphology, which is significant for male reproductive research. It also has significant potential for 3D dynamic observation of micro-organisms.

Fast measurement of surface defects on large components with dynamic phase-shifting digital holographic microscopy

The microscopic characterization of the surface defects on large opto-electronics components is essential to ensure the performance of major equipment. However, existing technology cannot achieve efficient and reliable fast cross-scale measurement due to the requirement on vertical scanning or polarization modulation for multi-frame capturing at each position. An effective approach is proposed to solve this problem, which needs to record only one interferometric hologram at each location and achieves full-area measurement by traversing over the surface. The overlapped area between adjacent regions are specified by wavefront registration and the topography in this area is obtained by two-step phase shifting and holographic reconstruction. Experimental results demonstrate that the proposed method can achieve fast and fast measurement of large specular components with high accuracy and high efficiency.

Phase-shifting digital holographic microscopy for microstructure measurement by sweeping the repetition rate of femtosecond laser

This paper proposes a phase-shifting digital holographic microscopy (PSDHM) for microstructure measurement by sweeping the repetition rate of femtosecond laser, and a multiple reflection arrangement between two quasi-parallel mirrors is constructed for optical multiplication. High precision phase-shifting can be achieved by sweeping the repetition rate of the femtosecond laser referenced to a Rb atomic clock without any mechanical sweeping. Optical multiplication can shorten the spatial distance of the optical delay line used for pulses alignment, make the PSDHM structure compact and stable, and avoid certain environmental disturbances. In the experiments, a ten-step phase-shifting test was first carried out for evaluating the phase-shifting accuracy, and the phase-shifting error was calculated to be in the range of −1° to 0.25°. Then, a USAF 1951 resolution target and a microstructure standard target were measured using a four-step PSDHM, and the measurement results were compared with those from a stylus profiler and a white light interferometer, respectively. The lateral resolution of PSDHM was tested to be about 2.1 μm, and the maximum error of the longitudinal measurement was within 6 nm. Experiments verify that the PSDHM system has good performance in terms of phase-shifting accuracy, surface topography measurement and coherent noise suppression.

Fast and pure phase-shifting off-axis holographic microscopy with a digital micromirror device.

We present a phase-shifting digital holographic microscopy technique, where a digital micromirror device enables to perform a precise phase-only shift of the reference wave. By coupling the beam into a monomode fiber, we obtain a laser mode with a constant phase shift, equally acting on all pixels of the hologram. This method has the advantage of being relatively simple and compatible with high frame rate cameras, which makes it of great interest for the observation of fast phenomena. We demonstrate the validity of the technique in an off-axis configuration by imaging living paramecia caudata.

Single-path single-shot phase-shifting digital holographic microscopy without a laser light source.

We propose single-path single-shot phase-shifting digital holographic microscopy (SSP-DHM) in which the quantitative phase information of an object wave is acquired without a laser light source. Multiple phase-shifted holograms are simultaneously obtained using a linear polarizer, a liquid crystal on a silicon spatial light modulator (LCoS-SLM), and a polarization-imaging camera. Complex amplitude imaging of a USAF1951 test target and phase imaging of transparent HeLa cells are performed to show its quantitative phase-imaging ability. We also conduct an experiment for the motion-picture imaging of transparent particles to highlight the single-shot imaging ability of SSP-DHM.

Characterization of Polarization Holographic Gratings Obtained on Azopolymer Thin Films by Digital Holographic Microscopy

Polarization diffraction gratings are formed by one-step polarization holographic recording in azopolymer thin films. The evolution of the gratings parameters, such as the modulation of diffraction efficiency and relief depth with regard to different exposure dose is analyzed. Phase-shifting digital holographic microscopy is applied for the measurement of the light-induced polarization diffraction gratings. For the accurate hologram acquisition and reconstruction of the complex amplitude transmitted by the gratings, we performed all-optical (without moving components) phase-shifting implemented within in the imaging system of the digital holographic microscope. The experimental measurement results and theoretical predictions were compared and analyzed.

Polarization holographic recording of vortex diffractive optical elements on azopolymer thin films and 3D analysis via phase-shifting digital holographic microscopy.

Direct fabrication of complex diffractive optical elements (DOEs) on photosensitive thin films is of critical importance for the development of advanced optical instruments. In this paper, we design and investigate DOEs capable of generating optical vortices. Analog and digital approaches for one-step polarization holographic recording of vortex DOEs on new carbazole-based azopolymer thin films are described. First configuration involves analog polarization holographic recording using a vortex phase retarder and has as a result the DOE producing a diffraction pattern with phase singularities aligned in a single line. Similar diffraction picture is achieved by the single-beam digital holographic recording setup with an integrated spatial light modulator. In the third system, the implemented double-beam digital polarization holographic recording setup yields simultaneously a spatial multiplexed vortex pattern. Diffraction efficiency evolution of these three types of DOEs are monitored and compared. The phase-shifting digital holographic microscope with an electrically controlled liquid crystal variable retarder is applied to investigate the phase and surface topography of the inscribed diffractive optical elements. The comparison between the digital and analog micro-patterning techniques contributes new evidence to limited data on the influence of the analog and digital generation of the spiral wavefront on the performance of vortex DOEs.

Improved 3D imaging of phase shifting digital holographic microscope by compensation for wavefront distortion

This paper is focused on improving the performance of quantitative phase imaging via phase-shifting digital holographic microscope. The development of the interferometric techniques is important for technology and biomedicine, since the surface and structure of samples can be monitored in real-time by non-destructive investigations. The novelty of the optical arrangement is that in the reference beam of the digital holographic microscope a liquid crystal variable retarder is introduced. Thus, it became possible to actively control the polarization state of light for realizing the necessary phase-shifts. In addition, a spatial light modulator is integrated in the optical setup to produce computer-controlled compensation of the spatial distortion. An ad hoc hologram processing technique was developed to execute the numerical correction of the physical phase-shifting errors. Topographical investigations of phase masks recorded on carbazol-based azopolymers provided the experimental testing of the achieved accuracy in phase reconstruction.

Modularized microscope based on parallel phase-shifting digital holography for imaging of living biospecimens.

.Significance: Parallel phase-shifting digital holographic microscope (PPSDHM) is powerful for three-dimensional (3D) measurements of dynamic specimens. However, the PPSDHM reported previously was directly fixed on the optical bench and imposed difficulties case, thus it is required to modify the specification of the microscope or transport the microscope to another location.Aim: We present a modularized PPSDHM. We construct the proposed PPSDHM and demonstrate the 3D measurement capability of the PPSDHM.Approach: The PPSDHM was designed as an inverted microscope to record transparent objects and modularized by integrating the optical elements of the PPSDHM on an optical breadboard. To demonstrate the effectiveness of the PPSDHM, we recorded a 3D motion-picture of moving Volvoxes at and carried out 3D tracking of the Volvoxes.Results: The PPSDHM was practically realized and 3D images of objects were successfully reconstructed from holograms recorded with a single-shot exposure. The 3D trajectories of Volvoxes were obtained from the reconstructed images.Conclusions: We established a modularized PPSDHM that is capable of 3D image acquisition by integrating the optical elements of the PPSDHM on an optical breadboard. The recording capability of 3D motion-pictures of dynamic specimens was experimentally demonstrated by the PPSDHM.

Study of low-noise phase-shifting digital holographic microscopy using a long working distance objective

In order to improve the measurement accuracy, a kind of phase shifting digital holographic microscopy based on a long working distance microscopic objective is proposed. In the setup, an LED is adopted as the illumination light source, which can suppress coherent noise effectively and hence improve the measurement accuracy. A michelson quasi-common-path interferometer is constructed by adding a beam-splitter between the long working distance objective and the sample. The layout of the setup is simple and it can be easily adjusted, and thus the interference can be come into being conveniently especially when the sample is illuminated with a partial light source. The blind phase-shifting interferometry is adopted in the reconstruction procedure, and the two-step blind phase-shifting algorithm is used to reconstruct the phase map of the measured sample. In the experiments, the height maps of a reflective USAF 1951 resolution target are measured under LED illumination and He-Ne laser illumination, respectively. The measurement results show that both coincide with each other; the phase noise under LED illumination is, however, reduced by 70% when compared with that under laser illumination. In addition, in order to further verify the effectiveness of the device, the device is used to measure a micro-nano rectangular step engraved on the silicon substrate. The measurement results are in good agreement with the nominal values. This technique can be potentially used in the topographic measurement of micro-structures.

Phase-shifting digital holographic microscopy with an iterative blind reconstruction algorithm.

In phase-shifting digital holographic microscopy (PS-DHM), the reconstructed phase map is obtained after processing several holograms of the same scene with a phase shift between them. Most of the reconstruction algorithms in PS-DHM require an accurate and known phase shift between the recorded holograms. This requirement limits the applicability of the method. To ease the use of PS-DHM, this paper presents an iterative-blind phase shift extraction method based on demodulation of the different components of the recorded holograms. The method uses a DHM system operating in a slightly off-axis architecture. The proposed method uses three-frame holograms with arbitrary and unequal phase shifts between them and therefore eases the use of the PS-DHM. We believe both simulated and experimental results demonstrate the goodness and feasibility of the proposed technique.

Common-path, single-shot phase-shifting digital holographic microscopy using a Ronchi ruling

Phase-shifting digital holography is widely considered to be a groundbreaking method to quantitatively investigate the phase distribution of specimens, such as living cells. The main flaws of this method, however, are that the requirement for several sequential phase-shifted holograms eliminates the possibility of single-shot imaging and complex configurations would also increase the temporal noise. The present paper aims to validate a single-shot, common-path, phase-shifting digital holographic microscopy, employing a self-referencing geometry. A Ronchi ruling, located in the Fourier plane of a standard microscopic imaging system, produces multiple replicas of sample information in the image plane. The phase retrieval algorithm is performed by superposition of the sample-free portion of each replica with the object information, and requires at least three adjacent diffraction orders. To evaluate the performance of the proposed method, the phase distribution of silica microspheres as a test sample and the morphology of red blood cells as a biological specimen are determined. This configuration offers improved temporal stability in comparison with previously reported Mach–Zehnder interferometers, and may serve as an alternative for real-time surveying of nanometric and subnanometric fluctuations of living microscopic specimens.

Three-dimensional refractive index measurement of special optical fiber based on optical vortex phase-shifting digital holographic microscopy

We propose an approach for three-dimensional (3-D) refractive index (RI) measurement of special optical fiber by phase-shifting digital holographic microscopy based on optical vortex. The experimental system consists of a modified Mach–Zehnder interferometer and related lab-developed software. The phase shifts are obtained by loading different computer-generated spiral phase holograms on a liquid-crystal spatial light modulator with digital blazed grating, which is more accurate than the traditional phase-shift methods. The optical vortex generated in +1 diffraction order of the diffraction field is utilized as the reference beam. Eight digital holograms with the phase-shift increments of 0, π / 2, π, and 3π / 2 are recorded by a CCD before and after the tested optical fiber is placed in the experimental system. Then, a four-step phase-shifting algorithm and phase unwrapping algorithm are adopted to extract the phase distribution from the holograms. Based on a filtered backprojection algorithm, the 3-D RI distribution of the tested fiber is obtained at high accuracy. Experimental results demonstrate the feasibility of the proposed method for 3-D RI measurement of special optical fiber.

Calibrated Phase-Shifting Digital Holographic Microscope Using a Sampling Moiré Technique

A calibrated phase-shifting digital holographic microscope system capable of improving the quality of reconstructed images is proposed. Phase-shifting errors are introduced in phase-shifted holograms for numerous reasons, such as the non-linearity of piezoelectric transducers (PZTs), wavelength fluctuations in lasers, and environmental disturbances, leading to poor-quality reconstructions. In our system, in addition to the camera used to record object information, an extra camera is used to record interferograms, which are used to analyze phase-shifting errors using a sampling Moiré technique. The quality of the reconstructed object images can be improved by the phase-shifting error compensation algorithm. Both the numerical simulation and experiment demonstrate the effectiveness of the proposed system.

Phase-shifting digital holographic microscopy by using a multi-camera setup.

In this Letter, the use of two-coupled Mach-Zehnder interferometers for four π/2-phase shifting interferometry is introduced. A multi-camera arrangement using no more than beam splitters and mirrors is utilized to obtain in a single shot the needed phase-shifted interferograms in the different output channels of the setup. The simplicity of the setup makes it ideal for high-speed interferometry applications. This proposal is validated in digital holographic microscopy to visualize a biological sample of epidermal onion cells.

Two-step phase shifting differential-recording digital holographic microscopy

We present two-step phase-shifting differential-recording digital holographic microscopy (TPD-DH in microscopy) for phase imaging of microscopic transparent elements. Two CCDs are employed to record two interferograms at two different defocusing distances. The interferograms on the two CCD cameras are shifted for a phase retarder 0 and π via an all-optics phase shifting unit. A novel algorithm is proposed to reconstruct both amplitude and phase distributions of the object wave from the recorded interferograms. This method has the same spectrum bandwidth and measurement accuracy with those of conventional four-step phase-shifting interferometry (FS-PSI), whereas it reduces the measurement time by half.

Phase measurement of structural modifications created by femtosecond laser pulses in glass with phase-shifting digital holographic microscopy

When ultrashort laser pulses are focused inside a glass at a high repetition rate, structural changes occur because of the heat accumulation from the train of laser pulses. This report describes phase measurement of the structural changes induced inside glass by phase-shifting digital holographic microscopy. Two-dimensional phase distribution across the structural change for static exposure is retrieved. By focusing femtosecond laser pulses at the interface between two glass substrates, melted materials can directly weld glass plates. Quantitative phase measurement of welded glass substrates revealed that the refractive index decreased in the laser-irradiated zone.

Computational out-of-focus imaging increases the space–bandwidth product in lens-based coherent microscopy

The space–bandwidth product (SBP) of modern objective lenses is often significantly larger than the pixel count of opto-electronic image sensor chips, and, therefore, much of the information transmitted by the optical system cannot be adequately sampled or digitized. To resolve this mismatch, microscopes are in general designed to maintain the resolution of the optical system while significantly wasting the field of view (FOV) and SBP of the objective lens. We introduce a wide-field and high-resolution coherent imaging method that uses a stack of out-of-focus images to provide much better utilization of the SBP of an objective lens. We demonstrate our approach on a benchtop microscope by using a demagnification camera adapter to match the active area of the image sensor chip to the FOV of an objective lens. We show that the resulting spatial undersampling caused by capturing a large FOV can be mitigated through an iterative pixel super-resolution algorithm that uses e.g., ∼three to five slightly out-of-focus images, yielding an ∼8-fold increase in the SBP of the microscope. Furthermore, the same pixel super-resolution algorithm also achieves phase retrieval, revealing the optical phase information of the specimen. We compared our method against traditional off-axis and phase-shifting digital holographic microscopy modalities and demonstrated at least 3-fold reduction in the number of images required to achieve the same SBP. This technique could be used to maximize the throughput and SBP of lens-based coherent imaging and holography systems and inspire new microscopy designs that benefit from the inherent autofocusing steps of a scanning microscope to increase its SBP.

Single-shot and phase-shifting digital holographic microscopy using a 2-D grating.

We demonstrate digital holographic microscopy that, while being based on phase-shifting interferometry, is capable of single-shot measurements. A two-dimensional (2-D) diffraction grating placed in a Fourier plane of a standard in-line holographic phase microscope generates multiple copies of a sample image on a camera sensor. The identical image copies are spatially separated with different overall phase shifts according to the diffraction orders. The overall phase shifts are adjusted by controlling the lateral position of the grating. These phase shifts are then set to be multiples of π/2. Interferograms composed of four image copies combined with a parallel reference beam are acquired in a single shot. The interferograms are processed through a phase-shifting algorithm to produce a single complex image. By taking advantage of the higher sampling capacity of the in-line holography, we can increase the imaging information density by a factor of 3 without compromising the imaging acquisition speed.

Phase-shifting by means of an electronically tunable lens: quantitative phase imaging of biological specimens with digital holographic microscopy.

The use of an electronically tunable lens (ETL) to produce controlled phase shifts in interferometric arrangements is shown. The performance of the ETL as a phase-shifting device is experimentally validated in phase-shifting digital holographic microscopy. Quantitative phase maps of a section of the thorax of a Drosophila melanogaster fly and of human red blood cells have been obtained using our proposal. The experimental results validate the possibility of using the ETL as a reliable phase-shifter device.