Is multiplexed off-axis holography for quantitative phase imaging more spatial bandwidth-efficient than on-axis holography? [Invited].

Digital holographic microcopy is a thriving imaging modality that attracts considerable research interest due to its ability not only to create excellent label-free contrast but also to supply valuable physical information regarding the density and dimensions of the sample with nanometer-scale axial sensitivity. Three basic holographic recording geometries currently exist, including on-axis, off-axis, and slightly off-axis holography, each of which enables a variety of architectures in terms of bandwidth use and compression capacity. Specifically, off-axis holography and slightly off-axis holography allow spatial hologram multiplexing, enabling one to compress more information into the same digital hologram. In this paper, we define an efficiency score to analyze the various possible architectures and compare the signal-to-noise ratio and the mean squared error obtained using each of them, thus determining the optimal holographic method.

Optimal spatial bandwidth capacity in multiplexed off-axis holography for rapid quantitative phase reconstruction and visualization: erratum.

We correct a typo that repeated itself in several equations. Our previous results and conclusions are unchanged.

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Four dimensional phase unwrapping of dynamic objects in digital holography.

We present a new four-dimensional phase unwrapping approach for time-lapse quantitative phase microscopy, which allows reconstruction of optically thick objects that are optically thin in a certain temporal point and angular view. We thus use all four dimensions of the dynamic quantitative phase profile acquired, including the angular dimension and the temporal dimension, in addition to the x-y dimensions. We first demonstrate the capabilities of this algorithm on simulative data, enabling the quantification of the reconstruction quality relative to both the ground truth and existing unwrapping approaches. Then, we demonstrate the applicability of the proposed four-dimensional phase unwrapping algorithm by experimentally capturing a dual-angular dynamic off-axis hologram with simultaneous recording of two angular views, using multiplexing of two off-axis holograms into a single multiplexed hologram.

Six-pack off-axis holography.

We present a new holographic concept, named six-pack holography (6PH), in which we compress six off-axis holograms into a single multiplexed off-axis hologram without loss of magnification or resolution. The multiplexed hologram contains straight off-axis fringes with six different orientations, and can be generated optically or digitally. We show that since the six different complex wavefronts do not overlap in the spatial frequency domain, they can be fully reconstructed. 6PH allows more than 50% improvement in the spatial bandwidth consumption when compared to the best multiplexing method proposed so far. We expect the 6PH concept to be useful for a variety of applications, such as field-of-view multiplexing, wavelength multiplexing, temporal multiplexing, multiplexing for super-resolution imaging, and others.

Angular phase unwrapping of optically thick objects with a thin dimension.

We present a new phase unwrapping approach, which allows reconstruction of optically thick objects that are optically thin from at least one viewing angle, by considering the information stored in the object phase maps captured from consecutive angles. Our algorithm combines 1-D phase unwrapping in the angular dimension with conventional 2-D phase unwrapping, to achieve unwrapping of the object from the optically thick perspective. We thus obtain quantitative phase imaging of objects that were previously impossible to image in certain viewing angles. To demonstrate our approach, we present both numerical simulation and experimental results for quantitative phase imaging of biological cells.

Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation.

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label-free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive-index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive-index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label-free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

Video-rate processing in tomographic phase microscopy of biological cells using CUDA.

We suggest a new implementation for rapid reconstruction of three-dimensional (3-D) refractive index (RI) maps of biological cells acquired by tomographic phase microscopy (TPM). The TPM computational reconstruction process is extremely time consuming, making the analysis of large data sets unreasonably slow and the real-time 3-D visualization of the results impossible. Our implementation uses new phase extraction, phase unwrapping and Fourier slice algorithms, suitable for efficient CPU or GPU implementations. The experimental setup includes an external off-axis interferometric module connected to an inverted microscope illuminated coherently. We used single cell rotation by micro-manipulation to obtain interferometric projections from 73 viewing angles over a 180° angular range. Our parallel algorithms were implemented using Nvidia's CUDA C platform, running on Nvidia's Tesla K20c GPU. This implementation yields, for the first time to our knowledge, a 3-D reconstruction rate higher than video rate of 25 frames per second for 256 × 256-pixel interferograms with 73 different projection angles (64 × 64 × 64 output). This allows us to calculate additional cellular parameters, while still processing faster than video rate. This technique is expected to find uses for real-time 3-D cell visualization and processing, while yielding fast feedback for medical diagnosis and cell sorting.

Interferometric phase microscopy for label-free morphological evaluation of sperm cells.

OBJECTIVE: To compare label-free interferometric phase microscopy (IPM) to label-free and label-based bright-field microscopy (BFM) in evaluating sperm cell morphology. This comparison helps in evaluating the potential of IPM for clinical sperm analysis without staining. DESIGN: Comparison of imaging modalities. SETTING: University laboratory. PATIENT(S): Sperm samples were obtained from healthy sperm donors. INTERVENTION(S): We evaluated 350 sperm cells, using portable IPM and BFM, according to World Health Organization (WHO) criteria. The parameters evaluated were length and width of the sperm head and midpiece; size and width of the acrosome; head, midpiece, and tail configuration; and general normality of the cell. MAIN OUTCOME MEASURE(S): Continuous variables were compared using the Student's t test. Categorical variables were compared with the χ(2) test of independence. Sensitivity and specificity of IPM and label-free BFM were calculated and compared with label-based BFM. RESULT(S): No statistical differences were found between IPM and label-based BFM in the WHO criteria. In contrast, IPM measurements of head and midpiece width and acrosome area were different from those of label-free BFM. Sensitivity and specificity of IPM were higher than those of label-free BFM for the WHO criteria. CONCLUSION(S): Label-free IPM can identify sperm cell abnormalities, with an excellent correlation with label-based BFM, and with higher accuracy compared with label-free BFM. Further prospective clinical trials are required to enable IPM as part of clinical sperm selection procedures.