Off-axis holographic multiplexing involves capturing several complex wavefronts, each encoded into off-axis holograms with different interference fringe orientations, simultaneously, with a single camera acquisition. Thus, the multiplexed off-axis hologram can capture several wavefronts at once, where each one encodes different information from the sample, using the same number of pixels typically required for acquiring a single conventional off-axis hologram encoding only one sample wavefront. This gives rise to many possible applications, with focus on acquisition of dynamic samples, with hundreds of scientific papers already published in the last decade. These include field-of-view multiplexing, depth-of-field multiplexing, angular perspective multiplexing for tomographic phase microscopy for 3-D refractive index imaging, multiple wavelength multiplexing for multiwavelength phase unwrapping or for spectroscopy, performing super-resolution holographic imaging with synthetic aperture with simultaneous acquisition, holographic imaging of ultrafast events by encoding different temporal events into the parallel channels using laser pulses, measuring the Jones matrix and the birefringence of the sample from a single multiplexed hologram, and measuring several fluorescent microscopy channels and quantitative phase profiles together, among others. Each of the multiplexing techniques opens new perspectives for applying holography to efficiently measure challenging biological and metrological samples. Furthermore, even if the multiplexing is done digitally, off-axis holographic multiplexing is useful for rapid processing of the wavefront, for holographic compression, and for visualization purposes. Although each of these applications typically requires a different optical system or processing, they all share the same theoretical background. We therefore review the theory, various optical systems, applications, and perspectives of the field of off-axis holographic multiplexing, with the goal of stimulating its further development.

Off-axis digital holographic multiplexing for rapid wavefront acquisition and processing

We demonstrate a successful deep learning strategy for cell identification and disease diagnosis using spatio-temporal cell information recorded by a digital holographic microscopy system. Shearing digital holographic microscopy is employed using a low-cost, compact, field-portable and 3D-printed microscopy system to record video-rate data of live biological cells with nanometer sensitivity in terms of axial membrane fluctuations, then features are extracted from the reconstructed phase profiles of segmented cells at each time instance for classification. The time-varying data of each extracted feature is input into a recurrent bi-directional long short-term memory (Bi-LSTM) network which learns to classify cells based on their time-varying behavior. Our approach is presented for cell identification between the morphologically similar cases of cow and horse red blood cells. Furthermore, the proposed deep learning strategy is demonstrated as having improved performance over conventional machine learning approaches on a clinically relevant dataset of human red blood cells from healthy individuals and those with sickle cell disease. The results are presented at both the cell and patient levels. To the best of our knowledge, this is the first report of deep learning for spatio-temporal-based cell identification and disease detection using a digital holographic microscopy system.

Deep learning-based cell identification and disease diagnosis using spatio-temporal cellular dynamics in compact digital holographic microscopy.

Microplastic fibers from synthetic textiles have been indicated as a major source of pollution because millions of fibers are released into the environment by laundry washing. In fact, such types of microplastics usually bypass wastewater treatment processes and filters, thus reaching the oceans and other water natural reservoirs in huge quantities. Nowadays, several approaches are available for characterizing microplastics, but unfortunately, there is no unique and standard method for this aim. Among the various methodologies, several microscopy techniques such as optical microscopy (OM), scanning electron microscopy coupled with energy dispersive X-ray (SEM–EDX) analysis, or OM combined with molecular spectroscopy can be used for visual detection and measurements. Our proposal is a non-destructive method based on a digital holographic microscope in a configuration fully sensitive to the polarization of light transmitted by the fibers with a micron dimension. Our aim is to prove that the proposed approach allows for the precise characterization of synthetic and natural fibers in water. The method exploits all the advantages of digital holography such as numerical refocusing, non-invasive testing, and quantitative measurements of the complex wave field by adding access to the polarization of light that conveys meaningful information about materials. We intend to show that a unique all-optical fingerprint can be retrieved using the Jones-matrix formalism for the major classes of synthetic microfilaments used in the textile industry (i.e., polyamide 6, polyamide 6.6, polypropylene, and polyester) and for the most common natural fibers (i.e., wool and cotton) prepared according to previously developed appropriate protocols. Our results show that the proposed technology identifies new features for micron-sized fibers based on optical anisotropy through quantitative digital holography that could open future routes for automatic and all-optical identification of textile contaminants in water. 

Toward an All-Optical Fingerprint of Synthetic and Natural Microplastic Fibers by Polarization-Sensitive Holographic Microscopy

Three-dimensional (3D) optical imaging of rapidly moving biological cells is difficult to achieve as such samples cannot be scanned over time. Here, we present a dynamic scan-free optical tomography approach for stain-free 3D imaging of biological cells using our new double six-pack tomography technique, whereby 12 off-axis holograms are captured in a single camera exposure without sacrificing resolution or field of view. The proposed system illuminates the sample from 12 angles simultaneously, and 3D refractive index (RI) tomograms are reconstructed from each recorded video frame of the dynamic sample. The technique is verified experimentally by recording flowing silica beads, 3 μm in diameter, with the resulting tomogram RI accuracy being 98.5%. A live swimming sperm cell is also imaged, and dynamic 3D imaging results for both beads and sperm cell are presented. The proposed technique represents a 12-fold increase in dynamic holographic data for tomography.

https://pubs.acs.org/doi/pdf/10.1021/acsphotonics.1c01804

Dynamic Tomographic Phase Microscopy by Double Six-Pack Holography

Birefringence is an important optical property of anisotropic materials arising from anisotropies of tissue microstructures. Birefringence parameters have been found to be important to understand optical anisotropic architecture of many materials and polarization imaging has been applied in many researches in the field of biology and medicine. Here, we propose a scheme to miniaturize a double-channel polarization holographic interferometer optics to create a polarization holographic microscope slide (P-HMS) suitable for integrating with microfluidic lab-on-a-chip (LoC) systems. Based on the P-HMS combined with a simple reconstruction algorithm described in the paper, we can not only simultaneously realize holographic imaging of two orthogonal polarization components of dynamic samples in a microfluidic channel but also quantitative measurement of 2D birefringence information, both including the birefringence phase retardation and optic-axis orientation. This chip interferometer allows for off-axis double-channel polarization digital holographic recording using only a single illumination beam without need of any beam splitter or mirror. Its quasi-common path configuration and self-aligned design also make it tolerant to vibrations and misalignment. This work about the P-HMS could play a positive role in promoting the application of birefringence imaging in microfluidic LoC technology.

Polarization holographic microscope slide for birefringence imaging of anisotropic samples in microfluidics

A compact system for single-shot sequential holographic imaging (SSSHI) with high temporal resolution and a large field of view is proposed. In this system, a specially designed sequence pulse train generator with a group of diffractive gratings inserted is adopted to simultaneously generate the probe pulse train and the reference pulse train required for recording a single-shot spatial frequency division multiplexing hologram. The system successfully overcomes the walk-off effect of the ultrashort pulse laser in SSSHI and, hence, effectively avoids the influence of the short coherence of ultrashort pulses on the spatial resolution (or field of view) of SSSHI; the complexity of the system and the difficulty in the precise synchronous alignment of the probe and the reference pulses also can be greatly reduced. An experimental setup of the system was constructed, and a SSSHI of dynamical air plasmas induced by a femtosecond pulse laser is successfully realized.

Single-shot ultrafast sequential holographic imaging with high temporal resolution and a large field of view

A method for realizing 2D single-shot measurements of birefringence parameters (including both retardation and optic axis orientation) of anisotropic materials using a simple recording setup and an efficient processing algorithm is proposed. The recording setup can be built simply by inserting a circular polarizer and a polarization beam splitter, respectively, in the object path and reference path of a conventional off-axis holographic imaging system, with no need for other adjustments. An algorithm for quantitatively retrieving the birefringence parameters from one single-shot hologram is proposed and demonstrated, in which a new quantity describing the birefringence, called complex birefringence parameter, is introduced, and a set of formulas used to extract the birefringence parameters is derived. Some experimental results are given for demonstrating the feasibility of the method that reveal that the method may provide another effective approach for investigating the birefringence properties of dynamic anisotropic materials, especially the birefringence induced by ultrafast pulse lasers.

Single-shot quantitative birefringence microscopy for imaging birefringence parameters.

Compact common-path polarisation holographic microscope for measuring spatially-resolved Jones matrix parameters of dynamic samples in microfluidics

The invention provides a single-strike sequence holographic imaging system and a single-strike sequence holographic imaging method. According to the system, a sequence pulse generator comprising a group of diffraction gratings is used for synchronously generating a detection pulse sequence and a reference pulse sequence required for realizing multichannel angle division multiplexing holographic recording, no included angle existing between the wavefronts of the two pulse sequences; and the detection pulse sequence and the reference pulse sequence enter a holographic recording unit. The walk-off effect of ultrashort pulse laser in the single-strike sequence holographic imaging system is overcome, and therefore, the limitation of the short coherence of ultrashort pulses on the spatial resolution and field of view of single-strike sequence holographic imaging is effectively avoided.

Hong-Yi Huang

Papers

Single-shot ultrafast sequential holographic imaging with high temporal resolution and a large field of view

Polarization holographic microscope slide for birefringence imaging of anisotropic samples in microfluidics

Single-shot quantitative birefringence microscopy for imaging birefringence parameters.

Compact common-path polarisation holographic microscope for measuring spatially-resolved Jones matrix parameters of dynamic samples in microfluidics

Single-strike sequence holographic imaging system and method