Off-axis Digital Holographic Microscopy Research Articles

High-throughput artifact-free slightly off-axis holographic imaging based on Fourier ptychographic reconstruction

Slightly off-axis digital holographic microscopy (DHM) has recently gained considerable attention due to its unique ability to improve the space-bandwidth product (SBP) of the imaging system while separating the object information from the background intensity to a certain extent. In order to obtain a decent image reconstruction, the spectral aliasing problem still needs to be addressed, which, however, is difficult to be achieved by the conventional linear Fourier domain filtering. To this end, in this paper, we propose a high-throughput artifact-free slightly off-axis holographic reconstruction method based on Fourier ptychographic microscopy (FPM). Inspired by the nonlinear optimized phase reconstruction algorithm of FPM, we perform constrained updates between the real and Fourier domains in an iterative manner to reconstruct the complex amplitude by the hologram intensity. Experimental results on live HeLa cell samples show that the proposed method can provide higher reconstruction accuracy and better image quality compared with the conventional Fourier method and the Kramers–Kronig (KK) relation-based method.

Interlaboratory evaluation of a digital holographic microscopy–based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.Graphical abstract

Compensation enhancement by the patch-based inpainting in off-axis digital holographic microscopy

Off-axis digital holographic microscopy is widely used to avoid the twin image problem in the hologram, which provides the advantages of extracting the real image. However, the off-axis optical setup and the microscope objective will bring tilt and spherical phase aberrations to the digital reconstruction process. Therefore, it is urgent to explore efficient techniques innovatively to compensate for these aberrations. This study proposes a numerical method via a patch-based inpainting algorithm to eliminate these aberrations. This method can reconstruct the actual object phase from a hologram without polynomial fitting. The reconstruction results in both the experiment and simulation are analyzed and compared, confirming that the proposed method has good robustness and accuracy in phase retrieval.

Spatial Consistency Calibration Based on Phase Difference Minimization for Parallel Slightly Off-Axis Digital Holographic Microscopy

In this study, we propose a spatial consistency calibration method for slightly off-axis digital holographic microscopy (SO-DHM). The acquisition of relative position errors is summarized into a solution of a nonlinear optimization problem in which the root mean squared error of phase aberration of holograms is minimized. The particle swarm optimization algorithm is chosen to solve this optimization problem due to its simple structure, high convergence efficiency, and robust global search ability. Phase-only wavefronts based on phase aberration, which remove the influence of noise, are used for calibration. The simulation result indicates that the proposed method has subpixel-level precision, and the experiment demonstrates the effectiveness of the proposed method in the SO-DHM system.

Digital holographic microscopy implementation for capillary filling measurements in nanoporous materials.

We report the implementation of lensless off-axis digital holographic microscopy as a non-destructive optical analyzer for nano-scale structures. The measurement capacity of the system was validated by analyzing the topography of a metallic grid with ≈150nm thick opaque features. In addition, an experimental configuration of self-reference was included to study the dynamics of the capillary filling phenomena in nanostructured porous silicon. The fluid front position as a function of time was extracted from the holograms, and the typical square root of time kinematics was recovered. The results shown are in agreement with previous works on capillary imbibition in similar structures and confirm a first step towards unifying holographic methods with fluid dynamics theory to develop a spatially resolved capillary tomography system for nanoporous materials characterization.

Compensation Enhancement by the Patch-Based Inpainting in Offaxis Digital Holographic Microscopy

Compensation Enhancement by the Patch-Based Inpainting in Offaxis Digital Holographic Microscopy

Video-Rate Quantitative Phase Imaging Using a Digital Holographic Microscope and a Generative Adversarial Network.

The conventional reconstruction method of off-axis digital holographic microscopy (DHM) relies on computational processing that involves spatial filtering of the sample spectrum and tilt compensation between the interfering waves to accurately reconstruct the phase of a biological sample. Additional computational procedures such as numerical focusing may be needed to reconstruct free-of-distortion quantitative phase images based on the optical configuration of the DHM system. Regardless of the implementation, any DHM computational processing leads to long processing times, hampering the use of DHM for video-rate renderings of dynamic biological processes. In this study, we report on a conditional generative adversarial network (cGAN) for robust and fast quantitative phase imaging in DHM. The reconstructed phase images provided by the GAN model present stable background levels, enhancing the visualization of the specimens for different experimental conditions in which the conventional approach often fails. The proposed learning-based method was trained and validated using human red blood cells recorded on an off-axis Mach–Zehnder DHM system. After proper training, the proposed GAN yields a computationally efficient method, reconstructing DHM images seven times faster than conventional computational approaches.

Adaptation of the Standard Off-Axis Digital Holographic Microscope to Achieve Variable Magnification

Traditional microscopy provides only for a small set of magnifications using a finite set of microscope objectives. Here, a novel architecture is proposed for quantitative phase microscopy that requires only a simple adaptation of the traditional off-axis digital holographic microscope. The architecture has the key advantage of continuously variable magnification, resolution, and Field-of-View, by simply moving the sample. The method is based on combining the principles of traditional off-axis digital holographic microscopy and Gabor microscopy, which uses a diverging spherical wavefield for magnification. We present a proof-of-concept implementation and ray-tracing is used to model the magnification, Numerical Aperture, and Field-of-View as a function of sample position. Experimental results are presented using a micro-lens array and shortcomings of the method are highlighted for future work; in particular, the problem of aberration is highlighted, which results from imaging far from the focal plane of the infinity corrected microscope objective.

Phase-aberration compensation via deep learning in digital holographic microscopy

Digital holographic microscopy (DHM), a quantitative phase-imaging technology, has been widely used in various applications. Phase-aberration compensation in off-axis DHM is vital to reconstruct topographical images with high precision, especially for microstructures with a small background or a dense phase distribution. We propose a numerical method based on deep learning combined with DHM. First, a convolutional neural network (CNN) recognizes and segments the sample and the background area of the hologram. Zernike polynomial fitting is then executed on the extracted background area. Finally, the whole process of phase-aberration compensation is automatically performed. To obtain a robust and accurate deep-learning model for hologram segmentation, we collected many holograms corresponding to several samples that had different morphological characteristics. The experimental results confirm that the trained CNN can accurately segment the sample from the background area of the hologram, and that this method is applicable and effective in off-axis DHM.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Autofocusing based on cosine similarity in dual-wavelength digital holographic microscopy

An autofocusing method based on cosine similarity in dual-wavelength digital holographic microscopy is proposed. In our experiments, red and green lasers are employed for illumination and the generated holograms are recorded by a color camera. During the refocusing process, the reconstructed optical field near the focus plane contains more regular features than the one on the defocus plane. Moreover, due to the wavelength dependency of the diffraction process, the further away from the focus plane, the greater the difference in the reconstructed optical fields from the two wavelengths. Therefore, the focus plane can be determined by finding the maximum value of the cosine similarity between the amplitude vectors from the reconstructed optical fields of the two wavelengths. Simulation and experimental results demonstrate the effectiveness of the proposed method in off-axis dual-wavelength digital holographic microscopy for shape measurement.

Off-Axis Digital Holographic Microscope with a Rotating Diffraction Plate for Speckle Suppression

Off-axis digital holographic microscopy (DHM) is a technique that can convert a single image, called hologram, into a 3D volume. A hologram encodes the phase and intensity information of the samples. Through post-processing, it can be used to reconstruct the quantitative phase map and track the spatial position of multiple samples. The temporal resolution is only limited by the acquisition speed of the camera. These unique features have been applied to studying sperm motility and bacterial dynamics. The phase information is encoded by the coherence fringes in the hologram and hence inevitably suffers from speckle noise of the laser. To suppress this speckle noise, we mounted a diffraction plate in front of the laser. The plate rotates at the velocity greater than the acquisition speed of the camera creating a time averaged hologram. The addition of the rotating diffraction plate significantly reduces the speckle noise, in turn mitigating errors during phase map reconstruction. We show here how this approach improves image quality and the reconstruction process and the ease with which it can be implemented in an open-frame DHM platform.

Adaptive spatial filtering based on fuzzy C-means and phase in off-axis digital holographic microscopy

Digital holographic microscopy can quantitatively image the biological samples label-free and noninvasively. It is key to extract the +1-term spectrum from the hologram spectrum, which is crucial to the quality of the reconstructed image. Therefore, an adaptive spatial filtering method based on fuzzy C-means and phase spectrum of a hologram is proposed to extract the +1-term spectrum without any prior knowledge. The maximum phase value point of phase spectrum is found, which must be located in the +1-term spectrum. Then, this point is first introduced to locate the +1-term spectrum region. Two classifications and three regions (+1-term, −1-term, and zero-order term spectra regions) are obtained by fuzzy C-means in the amplitude spectrum. Subsequently, the minimum distance between the centroids of the three regions and the maximum phase point is used to judge the +1-term spectrum region. Finally, a filtering window is obtained by the edge of the +1-term spectrum region and the +1-term spectrum is adaptively extracted. Compared to other spatial filtering methods, the proposed method avoids dependence on a prior custom mask and suppresses the higher frequency noise. Most importantly, the experimental results on a number of human cells and a phase step demonstrate the feasibility and efficiency of the proposed method.

Diffraction-based overlay metrology using angular-multiplexed acquisition of dark-field digital holograms.

In semiconductor device manufacturing, optical overlay metrology measures pattern placement between two layers in a chip with sub-nm precision. Continuous improvements in overlay metrology are needed to keep up with shrinking device dimensions in modern chips. We present first overlay metrology results using a novel off-axis dark-field digital holographic microscopy concept that acquires multiple holograms in parallel by angular multiplexing. We show that this concept reduces the impact of source intensity fluctuations on the noise in the measured overlay. With our setup we achieved an overlay reproducibility of 0.13 nm and measurements on overlay targets with known programmed overlay values showed good linearity of R2= 0.9993. Our data show potential for significant improvement and that digital holographic microscopy is a promising technique for future overlay metrology tools.

Automated extended depth of focus digital holographic microscopy using electrically tunable lens

A combination of electrically tunable lens with a microscope objective lens (MO) for multifocal plane imaging capability is proposed. The method aims at extending the depth of focus of the MO by changing its axial range and field of view. The combination is implemented in a common path off-axis digital holographic microscopy configuration experimentally and it achieves an axial scanning range of 363.5 μm. It utilizes image plane holography to obtain in-focus images throughout the axial range. The proposed method demonstrates its application in quantitative phase imaging of the USAF 1951 test chart and flowing red blood cells at different axial depths. An axial depth variation of 0.76 μm–0.81 μm is obtained between two consecutive focused planes. The results validate and postulate the advantage of the proposed method over traditional extended depth-of-focus algorithms. The quantitative and qualitative results exploit the possibility of using the proposed method further for localization and tracking of flowing microscopic samples.

Single-shot common-path off-axis dual-wavelength digital holographic microscopy

A single-shot common-path off-axis self-interference dual-wavelength digital holographic microscopic (DHM) system based on a cube beam splitter is demonstrated to expand the phase range in a stepped microstructure and for simultaneous measurement of the refractive index and physical thickness of a specimen. In the system, two laser beams with wavelengths of 532 nm and 632.8 nm are used. These laser beams are combined to transilluminate the object under study, then the object beam is divided into two beams by using a beam splitter oriented in such a way that both the beams propagate in almost the same direction, with an appropriate lateral separation between them. One of the object beams is spatially filtered at its Fourier plane, using a pinhole to generate a reference spherical beam free from the object information. The reference beam interferes with the object beam to form a digital hologram at the faceplate of the image sensor. The phase information is extracted from a single recorded digital hologram using the phase aberration compensation method that is based on principal component analysis (PCA). Owing to the common-path configuration, the system shows high temporal phase stability and it is less vibration-sensitive compared to counterparts such as a Mach-Zehnder type DHM. The performance of the dual-wavelength DHM system is verified in two different application fields by conducting the experiments using microsphere beads and living plant cells.

Axial displacement measurement with high resolution of particle movement based on compound digital holographic microscopy

In-line digital holographic particle imaging is gifted with high axial resolution but axial displacement of nearly-in-focus or in-focus particle is unable to be measured because of twin-image problem. Both in-focus and out-of-focus particles can be measured by off-axis digital holographic particle imaging but with poor axial resolution. These two make particle tracking in three-dimensional more complicated. This paper proposes a compound method of in-line and off-axis digital holographic microscopy to measure the axial displacement of both the in-focus and out-of-focus particle with improved resolution up to nanometer scale collectively. In the proposed compound digital holographic microscopy (CDHM), the critical reconstruction distance (CRD) is calculated. The twin image effect can be resolved by ensuring the reconstruction distance is larger than the CRD, then in-line digital holographic particle imaging can be effectively applied. If the reconstruction distance is smaller than the CRD, the ring pixels of optical path length (RPOPL) method is applied to improve resolution. The axial displacement of silica particles 5 μm and 10 μm in diameter fixed on the piezoelectric stage were successfully measured using CDHM method at resolution up to 4 nm (in-focus particle) and 2 nm (out-of-focus particle).

Digital Holographic Multimodal Cross-Sectional Fluorescence and Quantitative Phase Imaging System

We present a multimodal imaging system based on simple off-axis digital holography, for simultaneous recording and retrieval of cross-sectional fluorescence and quantitative phase imaging of the biological specimen. Synergism in the imaging capabilities can be achieved by incorporating two off-axis digital holographic microscopes integrated to record different information at the same time. The cross-sectional fluorescence imaging is realized by a common-path configuration of the single-shot off-axis incoherent digital holographic system. The quantitative phase imaging, on the other hand, is achieved by another off-axis coherent digital holographic microscopy operating in transmission mode. The fundamental characteristics of the proposed multimodal system are confirmed by performing various experiments on fluorescent beads and fluorescent protein-labeled living cells of the moss Physcomitrella patens lying at different axial depth positions. Furthermore, the cross-sectional live fluorescence and phase imaging of the fluorescent beads are demonstrated by the proposed multimodal system. The experimental results presented here corroborate the feasibility of the proposed system and indicate its potential in the applications to analyze the functional and structural behavior of biological cells and tissues.

Slightly off-axis digital holographic microscopy with a divided aperture

Divided aperture (DA) technology enhances the depth and anti-scattering capability in microscopy while slightly off-axis digital holographic microscopy improves the input-plane field of view (FOV) utilisation and optimises the detector measurement bandwidth. In this work, we propose a micro-imaging system utilising these two methods to perform full-field 3D topography measurements without any scanning. The DA eliminates the need for element tilts, pinholes, and gratings to produce an angle between the sample and reference beams. Two π/2-phase-shifted interferograms are obtained with polarising elements in one shot. Experiments are performed to prove the feasibility and validity of the proposed method.

Polarized Off-axis Digital Holographic Microscope with Partial Coherence Illumination

Off-axis digital holographic microscopy (DHM) is a well-known three-dimensional imaging method based on phase reconstruction. Lasers are typically favoured as the illumination source due to its long coherence length, which allows more tolerance between the object and reference paths. However, sub-reflections can cause interference and unwanted noise in the holograms. To solve this, Frank Dubois proposed a DHM with LED illumination due to its short coherence length [1]. However, the short coherence length requires extremely small differences between the two paths, which is difficult to achieve. Hence, to date LEDs have not been widely adopted in DHM.We present an off-axis DHM with a coherence enhanced LED, which leads to suppressed noise compared to a laser-based system. A pinhole was incorporated to strengthen the spatial coherence. Additionally, a bandpass filter was added to improve the temporal coherence resulting in an optimal coherence length. Polarized optics were used to facilitate the linear alignment of the beam path without sacrificing illumination power. We demonstrate the system's capabilities with both a standard resolution target and Hela cells.