Digital Holographic Microscopy System Research Articles

Common-path digital holographic microscopy based on a volume holographic grating for quantitative phase imaging.

Digital holographic microscopy (DHM) is a powerful quantitative phase imaging (QPI) technique that is capable of recording sample's phase information to enhance image contrast. In off-axis DHM, high-quality QPI images can be generated within a single recorded hologram, and the system stability can be enhanced by common-path configuration. Diffraction gratings are widely used components in common-path DHM systems; however, the presence of multiple diffraction beams leads to system power loss. Here, we propose and demonstrate implementation of a volume holographic grating (VHG) in common-path DHM, which provides single diffraction order. VHG in common-path DHM (i.e., VHG-DHM) helps in improving signal-to-noise ratio as compared to the conventional DHM. In addition, VHG, with inherently high angular selectivity, reduces image noise caused by stray light. With a simple fabrication process, it is convenient to utilize VHG to control the beam separation angle of DHM. Further, by using Bragg-matched wavelength degeneracy to avoid potential cell damaging effect in blue light, the VHG is designed for recording at a maximum sensitive wavelength of ∼488 nm, while our VHG-DHM is operated at the longer wavelength of red 632.8 nm for cell observation. Experimental results, measured by the VHG-DHM, show the measurement of target thickness ranging from 100 nm to 350 nm. In addition, stability of the system is quantitatively measured. High-contrast QPI images of human lung cancer cells are demonstrated.

Telecentric phase imaging at extended depth of focus using digital holographic microscopy

Digital holographic microscopy (DHM) is a well-known technique utilized by researchers to precisely examine the 3D topography of microscopic objects. Until now, DHM systems have offered researchers a trade-off between either achieving telecentricity or maintaining a limited depth of focus, presenting a challenge for comprehensive 3D imaging. Here, we propose a very stable DHM system that utilizes a customized microscope objective and electrically tunable lens to enable rapid axial scanning within the extended depth of focus range, continuous field of view, and telecentricity at each image plane. An extended depth of focus up to 5.5 mm axially is obtained with consistent magnification and resolution with a minimal magnification error of 2.2 % over the entire axial range. Additionally, a thorough investigation into the temporal stability of the system was conducted, with multiple holographic recordings taken over a period of 7 s. The minimal deviation in object height, measuring just 0.31 nm (without the use of vibration isolation), underscores the good temporal stability inherent in this proposed DHM system.

Live Cell Imaging by Single-Shot Common-Path Wide Field-of-View Reflective Digital Holographic Microscope.

Quantitative phase imaging by digital holographic microscopy (DHM) is a nondestructive and label-free technique that has been playing an indispensable role in the fields of science, technology, and biomedical imaging. The technique is competent in imaging and analyzing label-free living cells and investigating reflective surfaces. Herein, we introduce a new configuration of a wide field-of-view single-shot common-path off-axis reflective DHM for the quantitative phase imaging of biological cells that leverages several advantages, including being less-vibration sensitive to external perturbations due to its common-path configuration, also being compact in size, simple in optical design, highly stable, and cost-effective. A detailed description of the proposed DHM system, including its optical design, working principle, and capability for phase imaging, is presented. The applications of the proposed system are demonstrated through quantitative phase imaging results obtained from the reflective surface (USAF resolution test target) as well as transparent samples (living plant cells). The proposed system could find its applications in the investigation of several biological specimens and the optical metrology of micro-surfaces.

3D integral imaging of acoustically trapped objects

3D imaging provides crucial details about the objects and scenes that may not be obtained via 2D imaging methods. However, there are several applications in which the object to be 3D-imaged requires to be immobilized. The integrated digital holographic microscopy (DHM) and optical trapping (OT) system is a useful solution for such a task, but both DHM and OT are mostly suitable for microscopic specimens. Here, for the first time to the best of our knowledge and as an analogy to the DHM-OT system, we introduce integral imaging (InIm) and acoustic trapping (AT) integrated system for 3D imaging of immobilized mesoscopic and macroscopic objects. Post-processing of InIm data enables reconstructing the scene at any arbitrary plane, therefore, it re-focuses any particular depth of the object, which is a curtail task, especially when the object is trapped by AT. We demonstrate the capability of our system by simultaneous trapping and 3D imaging of single and multiple irregularly shaped objects with mm sizes.

In-focus quantitative phase imaging from defocused off-axis holograms: synergistic reconstruction framework.

Digital holographic microscopy (DHM) enables the three-dimensional (3D) reconstruction of quantitative phase distributions from a defocused hologram. Traditional computational algorithms follow a sequential approach in which one first reconstructs the complex amplitude distribution and later applies focusing algorithms to provide an in-focus phase map. In this work, we have developed a synergistic computational framework to compensate for the linear tilt introduced in off-axis DHM systems and autofocus the defocused holograms by minimizing a cost function, providing in-focus reconstructed phase images without phase distortions. The proposed computational tool has been validated in defocused holograms of human red blood cells and three-dimensional images of dynamic sperm cells.

Coded aperture temporal compressive digital holographic microscopy.

We report a coded aperture temporal compressive digital holographic microscopy (CATCHY) system to capture high-speed high-resolution samples by integrating snapshot compressive imaging (SCI) into digital holographic microscopy. Specifically, a two-dimensional (2D) detector samples a 4D (x, y, z, t) spatiotemporal data in a compressive manner, and after this, an efficient deep learning-based video SCI reconstruction algorithm is employed to reconstruct the desired 4D data cube. Up to ten high-resolution microscopic images are reconstructed from a snapshot measurement captured by our CATCHY system. Experimental results demonstrate the potential to visualize the 3D dynamic process of micro-nanostructures and imaging biological samples with high spatial and temporal resolution.

Comprehensive tool for a phase compensation reconstruction method in digital holographic microscopy operating in non-telecentric regime.

Quantitative phase imaging (QPI) via Digital Holographic microscopy (DHM) has been widely applied in material and biological applications. The performance of DHM technologies relies heavily on computational reconstruction methods to provide accurate phase measurements. Among the optical configuration of the imaging system in DHM, imaging systems operating in a non-telecentric regime are the most common ones. Nonetheless, the spherical wavefront introduced by the non-telecentric DHM system must be compensated to provide undistorted phase measurements. The proposed reconstruction approach is based on previous work from Kemper's group. Here, we have reformulated the problem, reducing the number of required parameters needed for reconstructing phase images to the sensor pixel size and source wavelength. The developed computational algorithm can be divided into six main steps. In the first step, the selection of the +1-diffraction order in the hologram spectrum. The interference angle is obtained from the selected +1 order. Secondly, the curvature of the spherical wavefront distorting the sample's phase map is estimated by analyzing the size of the selected +1 order in the hologram's spectrum. The third and fourth steps are the spatial filtering of the +1 order and the compensation of the interference angle. The next step involves the estimation of the center of the spherical wavefront. An optional final optimization step has been included to fine-tune the estimated parameters and provide fully compensated phase images. Because the proper implementation of a framework is critical to achieve successful results, we have explicitly described the steps, including functions and toolboxes, required for reconstructing phase images without distortions. As a result, we have provided open-access codes and a user interface tool with minimum user input to reconstruct holograms recorded in a non-telecentric DHM system.

The phase range extension and accuracy improvement in Fresnel biprism-based digital holography microscopy

We report a highly stable and affordable dual-wavelength digital holographic microscopy system based on common-path geometry. A Fresnel biprism is used to create an off-axis geometry, and two diode laser sources with different wavelengths λ1 = 532nm and λ2 = 650nm generate the dual-wavelength compound hologram. In order to extend the measurement range, the phase distribution is obtained using a synthetic wavelength Λ1 = 2930.5nm. Furthermore, to improve the system's temporal stability and reduce speckle noise, a shorter wavelength (Λ2 = 292.5nm) is used. The feasibility of the proposed configuration is validated by the experimental results obtained with Molybdenum trioxide, Paramecium, and red blood cell specimens.

Robust and compact digital Lensless Holographic microscope for Label-Free blood smear imaging

The lack of equipped healthcare infrastructure in isolated hard-to-reach zones exposes their population to a higher risk of complications in common diseases. With a timely diagnosis setting a life-altering difference, worldwide efforts have been conducted for the development of point-of-care testing (PoCT) with cost-effective devices. Among the most common interests in PoCT is the analysis of blood smear samples, as they can help to detect, diagnose, and monitor a wide range of diseases and disorders. With microscopy being the traditional tool for these analyses, a significative advance has been the development of cost-effective digital holographic microscopy systems, driven in part by its label-free imaging capabilities that waive the need for any sample preprocessing. Here, a robust and portable digital lensless holographic microscope, functionalized for the analysis of non-preprocessed blood smear samples in PoCT environments, is presented, and its viability is tested in the observation of red blood cells. The device uses an optical fiber with a cone-shaped tip instead of a pinhole, which ensures the sturdiness of the system and eliminates the need for challenging alignment. While the distances of the microscope can be tuned before fabrication, the herein-reported operational parameters are functionalized for the specific analysis of blood samples.

DHM-viewer: Comprehensive, open-source image processing suite for digital holographic microscopy.

In recent years, digital holographic microscopy (DHM) has witnessed extensive use in quantitative phase imaging (QPI) applications such as live cell dynamics imaging, and 3D particle tracking owing to its ability to reconstruct optical fields based on a single exposure. As a computational imaging technique, DHM is highly specific to the processing pipeline depending on optical configuration and processing requirements. Commercial DHM systems additionally rely on proprietary user interfaces and specific workflows that prohibit user modification, limiting their appeal for users who wish to explore different optical configurations or implement the latest processing algorithms. To address these demands, we present a comprehensive open-source software suite (DHM-viewer) compatible with most commercially available and bespoke DHM systems. DHM-viewer provides user-facing functionalities such as optical alignment assistance, stage actuation control, real-time live image acquisition, and post-processing visualization with a Graphical User Interface (GUI) system. Notably, the GUI is driven by a Standard Operation Procedure (SOP) processing layout that assist users and can accommodate various optical configurations and operating modes. From a system control perspective, DHM-viewer exploits the Micro-Manager framework to ensure compatibility with many existing microscope automation toolkits. From a post-processing perspective, the core software supports a variety of state-of-the-art phase aberration correction and Fourier filtering algorithms as well as deep learning-accelerated phase unwrapping and 3D tracking functionality leveraging the computational power of GPU-based processing. Finally, the DHM-viewer backend supports large batch offline processing through dedicated server-side distribution. The functionality and usability of the software were verified on three different DHM platforms, including one hybrid DHM-fluorescence system.

Combining off-axis digital holography with fluorescence for simultaneous fluorescence and quantitative phase imaging.

Off-axis digital holographic microscopy (DHM) is a microscopy technique capable of extracting and translating quantitative phase information (QPI) from the sample into 4D topographical data, which enables the study of phenotypic changes with sub-second temporal resolution. DHM has been widely implemented for long-term live-cell imaging and microelectromechanical systems characterization. However, as a bright field technique, relying on DHM imaging alone can be challenging especially in characterizing sub-cellular structures. Here we report on the design and implementation of a DHM module that can be retrofitted into a commercial dual-deck Olympus IX83 microscope base, transforming the inverted microscope into a hybrid DHM and fluorescence microscopy (DHM-FM) system. In order to achieve simultaneous acquisition of DHM and fluorescence data, the DHM module is designed to integrate illumination, dichroic filter, and interference submodules. The condenser of the microscope was replaced by the DHM illumination module, and the dichroic module was placed in the microscope platform to separate the DHM beam from the fluorescence signal. Finally, the DHM signal was delivered to the interference module outside the microscope body. As the DHM and fluorescence subsystems share the same field of view, this platform can acquire complementary data simultaneously. The capabilities of this coupled platform were explored through a series of pilot studies, including examining phase condensate formation and cellular dynamics. This modular configuration will help others explore the potential of off-axis DHM in their own work and, in particular, the potential of coupled DHM-fluorescence imaging.

Measurement of liquid refractive index by quantitative phase reconstruction of single frame dual-wavelength digital hologram

A measurement method of liquid’s refractive index by means of single-shot dual-wavelength off-axis digital holographic microscopy system and a microfluidic chip is presented. The refractive index of liquid filled in the microchannel of a microfluidic chip can be calculated directly with the unwrapped phase map obtained at the synthetized wavelength, without requirement of knowing another liquid’s refractive index as a measurement reference. Moreover, it also avoids the influence of the accuracy of the known refractive index on the refractive index to be measured. The refractive indices of salt solutions and sucrose solutions in the several concentrations are achieved in the experiments, which verifies that the measurement of liquid’s refractive index is accurate and reliable. With the merit of accurate measurement of liquid’s refractive index based on the one-frame dual-wavelength hologram, it is expected that this approach will be employed to detect in real-time the fluid’s parameters related to its refractive index.

A Novel Approach for Measurement of Free-Form Optical Elements with Digital Holographic Microscopy.

Free-form optical elements face significant challenges in high-precision measurement due to their high complexity and non-rotational symmetry. Digital holographic microscopy (DHM), as one of the methods for the measurement of free-form optical elements, has promising applications due to its ultra-high precision and non-destructive and fast characteristics. Therefore, we have designed a novel measurement method that combines transmission DHM and reflection DHM to obtain thickness information and surface information of elements to deduce the 3D structure. With this method, we completed the measurement of a free-form optical element. The DHM system we built has recorded holograms under 4× and 20× objectives and successfully recovered the 3D surface shape of the element. The measurements are consistent with the designed and manufactured parameters, demonstrating the unique advantages of DHM for measuring special types of optical elements.

Real-time 3D imaging of ocean algae with crosstalk suppressed single-shot digital holographic microscopy.

Digital holographic microscopy (DHM) has the potential to reconstruct the 3D shape of volumetric samples from a single-shot hologram in a label-free and noninvasive manner. However, the holographic reconstruction is significantly compromised by the out-of-focus image resulting from the crosstalk between refocused planes, leading to the low fidelity of the results. In this paper, we propose a crosstalk suppression algorithm-assisted 3D imaging method combined with a home built DHM system to achieve accurate 3D imaging of ocean algae using only a single hologram. As a key step in the algorithm, a hybrid edge detection strategy using gradient-based and deep learning-based methods is proposed to offer accurate boundary information for the downstream processing. With this information, the crosstalk of each refocused plane can be estimated with adjacent refocused planes. Empowered by this method, we demonstrated successful 3D imaging of six kinds of ocean algae that agree well with the ground truth; we further demonstrated that this method could achieve real-time 3D imaging of the quick swimming ocean algae in the water environment. To our knowledge, this is the first time single-shot DHM is reported in 3D imaging of ocean algae, paving the way for on-site monitoring of the ocean algae.

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

Linearity and Optimum-Sampling in Photon-Counting Digital Holographic Microscopy

In the image plane configurations frequently used in digital holographic microscopy (DHM) systems, interference patterns are captured by a photo-sensitive array detector located at the image plane of an input object. The object information in these patterns is localized and thus extremely sensitive to phase errors caused by nonlinear hologram recordings (grating profiles are either square or saturated sinusoidal) or inadequate sampling regarding the information coverage (undersampled around the Nyquist frequency or arbitrarily oversampled). Here, we propose a solution for both hologram recording problems through implementing a photon-counting detector (PCD) mounted on a motorized XY translation stage. In such a way, inherently linear (because of a wide dynamic range of PCD) and optimum sampled (due to adjustable steps) digital holograms in the image plane configuration are recorded. Optimum sampling is estimated based on numerical analysis. The validity of the proposed approach is confirmed experimentally.

Integrated self-referencing single shot digital holographic microscope and optical tweezer

Digital holographic microscopy is a single-shot technique for quantitative phase imaging of samples, yielding thickness profiles of phase objects. It provides sample features based on their morphology, leading to their classification and identification. However, observing samples, especially cells, in fluids using holographic microscopes is difficult without immobilizing the object. Optical tweezers can be used for sample immobilization in fluids. The present manuscript provides an overview of our ongoing work on the development of a compact, low-cost microscopy system for digital holographic imaging of optically trapped samples. Integration of digital holographic microscopy system with tweezers is realized by using the optical pickup unit extracted from DVD burners to trap microsamples, which are then holographically imaged using a highly compact self-referencing interferometer along with a low-cost, in-house developed quadrant photodiode, providing morphological and spectral information of trapped particles. The developed integrated module was tested using polystyrene microspheres as well as human erythrocytes. The investigated system offers a multitude of sample features, including physical and mechanical parameters and corner frequency information of the sample. These features were used for sample classification. The proposed technique has vast potential in opening up new avenues for low-cost, digital holographic imaging and analysis of immobilized samples in fluids and their classification.

Quantitative dynamic evolution of physiological parameters of RBC by highly stable digital holographic microscopy

Digital holographic microscopy (DHM) is a powerful label-free imaging tool that provides three-dimensional (3D) quantitative information of a specimen. In this work, the evaluation of morphological and quantitative parameters of human red blood cells (RBCs) by a new single-shot common-path off-axis digital holographic microscopic system is demonstrated. The proposed system is accomplished by employing a wedge plat, silver-coated at its back surface, into the object beam path generating two beams: one from the front surface and another from the back surface of the wedge plate. One of the beams is spatially filtered by using a pinhole to completely erase the object information from it and serving the clean reference beam, which on interfering with the object beam, creates the hologram. The proposed system, owing to common-path configuration, offers higher temporal phase stability, therefore, making it more suitable for the investigation of small cell thickness fluctuation. Moreover, the system is simple, compact, less expensive, and less vibration sensitive. The measurements of morphological and quantitative parameters, and membrane fluctuations of the human RBCs by the proposed system are reported. The experimentally calculated parameters of the RBC are obtained in good agreement with their normal physiological range.

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.

A common-path digital holographic microscope with a rotatable birefringent prism for synthesizing high-quality quantitative phase images from multi-angle interferograms

We propose a simple common-path off-axis interferometric system to perform high-quality quantitative phase imaging based on a rotatable birefringent prism that is placed at the output port of a conventional transmission or reflection microscope. The proposed system effectively eliminates the zero-order term of holographic data and increases the phase image reconstruction bandwidth by compounding multi-angle holograms, thereby obtaining high-quality quantitative phase images without zero-order artifacts. In addition, the proposed multi-angle phase image compounding method can eliminate the repetitive object phases caused by self-reference interferometry, thereby alleviating the problem of field-of-view reduction. Furthermore, we realize high-quality quantitative phase imaging based on multi-angle phase images without sacrificing the imaging frame rate. The concept is demonstrated by performing high-quality quantitative phase imaging of polystyrene microspheres and a freshwater alga, Pinnularia. Experimental results show that the noise level of the quantitative phase images synthesized using the proposed rotatable common-path digital holographic microscopy system is lower than that of conventional single-shot digital holographic microscope reconstruction quantitative images, and a more detailed cell internal structure can be observed.