Imaging biological tissue with high-throughput single-pixel compressive holography.

Single-pixel holography (SPH) is capable of generating holographic images with rich spatial information by employing only a single-pixel detector. Thanks to the relatively low dark-noise production, high sensitivity, large bandwidth, and cheap price of single-pixel detectors in comparison to pixel-array detectors, SPH is becoming an attractive imaging modality at wavelengths where pixel-array detectors are not available or prohibitively expensive. In this work, we develop a high-throughput single-pixel compressive holography with a space-bandwidth-time product (SBP-T) of 41,667 pixels/s, realized by enabling phase stepping naturally in time and abandoning the need for phase-encoded illumination. This holographic system is scalable to provide either a large field of view (~83 mm2) or a high resolution (5.80 µm × 4.31 µm). In particular, high-resolution holographic images of biological tissues are presented, exhibiting rich contrast in both amplitude and phase. This work is an important step towards multi-spectrum imaging using a single-pixel detector in biophotonics.

Using Augmented Reality to Reduce Fear and Promote Cooperation During Pediatric Otolaryngologic Procedures.

This case series examines interactive AR during minor otolaryngologic procedures. Although VR has been successfully used for pediatric vascular access, removing children from comforting people in the real world has resulted in patient anxiety. AR offers a potential advantage, utilizing distracting holographic images when patients maintain eye contact with parents. The primary objective was to determine the effect of AR on fear during pediatric otolaryngologic procedures. Secondary objectives included evaluating pain; procedure compliance; and patient, parent and physician attitudes toward AR, as well as assessing the feasibility of adding AR to a busy outpatient otolaryngologic clinic. Laryngoscope, 131:E1342-E1344, 2021.

Deep learning-based super-resolution in coherent imaging systems.

We present a deep learning framework based on a generative adversarial network (GAN) to perform super-resolution in coherent imaging systems. We demonstrate that this framework can enhance the resolution of both pixel size-limited and diffraction-limited coherent imaging systems. The capabilities of this approach are experimentally validated by super-resolving complex-valued images acquired using a lensfree on-chip holographic microscope, the resolution of which was pixel size-limited. Using the same GAN-based approach, we also improved the resolution of a lens-based holographic imaging system that was limited in resolution by the numerical aperture of its objective lens. This deep learning-based super-resolution framework can be broadly applied to enhance the space-bandwidth product of coherent imaging systems using image data and convolutional neural networks, and provides a rapid, non-iterative method for solving inverse image reconstruction or enhancement problems in optics.

ConoSurf: Open-source 3D scanning system based on a conoscopic holography device for acquiring surgical surfaces.

BACKGROUND: A difficulty in computer-assisted interventions is acquiring the patient's anatomy intraoperatively. Standard modalities have several limitations: low image quality (ultrasound), radiation exposure (computed tomography) or high costs (magnetic resonance imaging). An alternative approach uses a tracked pointer; however, the pointer causes tissue deformation and requires sterilizing. Recent proposals, utilizing a tracked conoscopic holography device, have shown promising results without the previously mentioned drawbacks. METHODS: We have developed an open-source software system that enables real-time surface scanning using a conoscopic holography device and a wide variety of tracking systems, integrated into pre-existing and well-supported software solutions. RESULTS: The mean target registration error of point measurements was 1.46 mm. For a quick guidance scan, surface reconstruction improved the surface registration error compared with point-set registration. CONCLUSIONS: We have presented a system enabling real-time surface scanning using a tracked conoscopic holography device. Results show that it can be useful for acquiring the patient's anatomy during surgery.

Portable waveguide display system with a large field of view by integrating freeform elements and volume holograms.

A compact waveguide display system integrating freeform elements and volume holograms is presented here for the first time. The use of freeform elements can broaden the field of view, which limits the applications of a holographic waveguide. An optimized system can achieve a diagonal field of view of 45° when the thickness of the waveguide planar is 3mm. Freeform-elements in-coupler and the volume holograms out-coupler were designed in detail in our study, and the influence of grating configurations on diffraction efficiency was analyzed thoroughly. The off-axis aberrations were well compensated by the in-coupler and the diffraction efficiency of the optimized waveguide display system could reach 87.57%. With integrated design, stability and reliability of this monochromatic display system were achieved and the alignment of the system was easily controlled by the record of the volume holograms, which makes mass production possible.

Improvement of gray-scale representation of horizontally scanning holographic display using error diffusion.

Horizontally scanning holography can enlarge both screen size and viewing zone angle. A microelectromechanical-system spatial light modulator, which can generate only binary images, is used to generate hologram patterns. Thus, techniques to improve gray-scale representation in reconstructed images should be developed. In this study, the error diffusion technique was used for the binarization of holograms. When the Floyd-Steinberg error diffusion coefficients were used, gray-scale representation was improved. However, the linearity in the gray-scale representation was not satisfactory. We proposed the use of a correction table and showed that the linearity was greatly improved.

Real-time quantitative phase reconstruction in off-axis digital holography using multiplexing.

We present a new approach for obtaining significant speedup in the digital processing of extracting unwrapped phase profiles from off-axis digital holograms. The new technique digitally multiplexes two orthogonal off-axis holograms, where the digital reconstruction, including spatial filtering and two-dimensional phase unwrapping on a decreased number of pixels, can be performed on both holograms together, without redundant operations. Using this technique, we were able to reconstruct, for the first time to our knowledge, unwrapped phase profiles from off-axis holograms with 1 megapixel in more than 30 frames per second using a standard single-core personal computer on a MATLAB platform, without using graphic-processing-unit programming or parallel computing. This new technique is important for real-time quantitative visualization and measurements of highly dynamic samples and is applicable for a wide range of applications, including rapid biological cell imaging and real-time nondestructive testing. After comparing the speedups obtained by the new technique for holograms of various sizes, we present experimental results of real-time quantitative phase visualization of cells flowing rapidly through a microchannel.

Common-path configuration in total internal reflection digital holography microscopy.

Total Internal Reflection Digital Holographic Microscopy (TIRDHM) is recognized to be a powerful tool for retrieving quantitative phase images of cell-substrate interfaces, adhesions, and tissue structures close to the prism surface. In this Letter, we develop an improved TIRDHM system, taking advantage of a refractive index mismatch between the prism and the sample substrate, to allow phase-shifting DH with just a single-beam interferometric configuration. Instead of the traditional off-axis method, phase-shift method is used to retrieve amplitude and phase images in coherent light and TIR modality. Essentially, the substrate-prism interface acts like a beam splitter generating a reference beam, where the phase-shift dependence on the incident angle is exploited in this common-path configuration. With the aim to demonstrate the technique's validity, some experiments are performed to establish the advantage of this compact and simple configuration, in which the reference arm in the setup is avoided.

A comparison study of different facial soft tissue analysis methods.

OBJECTIVES: The purpose of this study was to evaluate several different facial soft tissue measurement methods. MATERIALS AND METHODS: After marking 15 landmarks in the facial area of 12 mannequin heads of different sizes and shapes, facial soft tissue measurements were performed by the following 5 methods: Direct anthropometry, Digitizer, 3D CT, 3D scanner, and DI3D system. With these measurement methods, 10 measurement values representing the facial width, height, and depth were determined twice with a one week interval by one examiner. These data were analyzed with the SPSS program. RESULTS: The position created based on multi-dimensional scaling showed that direct anthropometry, 3D CT, digitizer, 3D scanner demonstrated relatively similar values, while the DI3D system showed slightly different values. All 5 methods demonstrated good accuracy and had a high coefficient of reliability (>0.92) and a low technical error (<0.9 mm). The measured value of the distance between the right and left medial canthus obtained by using the DI3D system was statistically significantly different from that obtained by using the digital caliper, digitizer and laser scanner (p < 0.05), but the other measured values were not significantly different. On evaluating the reproducibility of measurement methods, two measurement values (Ls-Li, G-Pg) obtained by using direct anthropometry, one measurement value (N'-Prn) obtained by using the digitizer, and four measurement values (EnRt-EnLt, AlaRt-AlaLt, ChRt-ChLt, Sn-Pg) obtained by using the DI3D system, were statistically significantly different. However, the mean measurement error in every measurement method was low (<0.7 mm). All measurement values obtained by using the 3D CT and 3D scanner did not show any statistically significant difference. CONCLUSION: The results of this study show that all 3D facial soft tissue analysis methods demonstrate favorable accuracy and reproducibility, and hence they can be used in clinical practice and research studies.

Common path in-line holography using enhanced joint object reference digital interferometers.

Joint object reference digital interferometer (JORDI) is a recently developed system capable of recording holograms of various types [Opt. Lett. 38(22), 4719 (2013)]. Presented here is a new enhanced system design that is based on the previous JORDI. While the previous JORDI has been based purely on diffractive optical elements, displayed on spatial light modulators, the present design incorporates an additional refractive objective lens, thus enabling hologram recording with improved resolution and increased system applicability. Experimental results demonstrate successful hologram recording for various types of objects, including transmissive, reflective, three-dimensional, phase and highly scattering objects. The resolution limit of the system is analyzed and experimentally validated. Finally, the suitability of JORDI for microscopic applications is verified as a microscope objective based configuration of the system is demonstrated.

On artefact-free reconstruction of low-energy (30-250eV) electron holograms.

Low-energy electrons (30-250eV) have been successfully employed for imaging individual biomolecules. The most simple and elegant design of a low-energy electron microscope for imaging biomolecules is a lensless setup that operates in the holographic mode. In this work we address the problem associated with the reconstruction from the recorded holograms. We discuss the twin image problem intrinsic to inline holography and the problem of the so-called biprism-like effect specific to low-energy electrons. We demonstrate how the presence of the biprism-like effect can be efficiently identified and circumvented. The presented sideband filtering reconstruction method eliminates the twin image and allows for reconstruction despite the biprism-like effect, which we demonstrate on both, simulated and experimental examples.

Three-dimensional identification of microorganisms using a digital holographic microscope.

This paper reports a method for three-dimensional (3D) analysis of shift-invariant pattern recognition and applies to holographic images digitally reconstructed from holographic microscopes. It is shown that the sequential application of a 2D filter to the plane-by-plane reconstruction of an optical field is exactly equivalent to the application of a more general filter with a 3D impulse response. We show that any 3D filters with arbitrary impulse response can be implemented in this way. This type of processing is applied to the two-class problem of distinguishing different types of bacteria. It is shown that the proposed technique can be easily implemented using a modified microscope to develop a powerful and cost-effective system with great potential for biological screening.

Automated segmentation of multiple red blood cells with digital holographic microscopy.

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

Accuracy and reproducibility of 3-dimensional digital model measurements.

INTRODUCTION: The purpose of this study was to evaluate the reliability of measurements made on 3-dimensional digital models obtained with a surface laser scanner (D-250; 3Shape, Copenhagen, Denmark). METHODS: Twenty orthodontic dental casts of permanent dentitions were selected. Three-dimensional images were obtained on this scanner and analyzed by using the Geomagic Studio 5 software (Raindrop Geomagic, Inc, Morrisville, NC). Measurements were made with a digital caliper directly on the dental casts and also digitally on the digital models. Fifteen anatomic dental points were identified, and a total of 11 linear measurements were taken from each cast, including arch length and width. Dependent t tests were used to evaluate intraexaminer reproducibility and measurement accuracy on the digital models. RESULTS: No statistically significant differences were found between the measurements made directly on the dental casts and on the digital models. CONCLUSIONS: Linear measurements on digital models are accurate and reproducible. Digital models obtained with the surface laser scanner are reliable for measurements of arch width and length.

Quantifying cellular differentiation by physical phenotype using digital holographic microscopy.

Although the biochemical changes that occur during cell differentiation are well-known, less known is that there are significant, cell-wide physical changes that also occur. Understanding and quantifying these changes can help to better understand the process of differentiation as well as ways to monitor it. Digital holographic microscopy (DHM) is a marker-free quantitative phase microscopy technique for measuring biological processes such as cellular differentiation, alleviating the need for introduction of foreign markers. We found significant changes in subcellular structure and refractive index of differentiating myeloid precursor cells within one day of differentiation induction, and significant differences depending on the type of lineage commitment. We augmented our results by showing significant changes in the softness of myeloid precursor cell differentiation within one day using optical stretching, a laser trap-based marker-free technique. DHM and optical stretching therefore provide consequential parameterization of cellular differentiation with sensitivity otherwise difficult to achieve. Therefore, we provide a way forward to quantify and understand cell differentiation with minimal perturbation using biophotonics.

Beyond the lateral resolution limit by phase imaging.

We present a theory to extend the classical Abbe resolution limit by introducing a spatially varying phase into the illumination beam of a phase imaging system. It allows measuring lateral and axial distance differences between point sources to a higher accuracy than intensity imaging alone. Various proposals for experimental realization are debated. Concretely, the phase of point scatterers' interference is experimentally visualized by high numerical aperture (NA = 0.93) digital holographic microscopy combined with angular scanning. Proof-of-principle measurements are presented by using sub-wavelength nanometric holes on an opaque metallic film. In this manner, Rayleighs classical two-point resolution condition can be rebuilt. With different illumination phases, enhanced bandpass information content is demonstrated, and its spatial resolution is theoretically shown to be potentially signal-to-noise ratio limited.

Effects of aliasing on the fidelity of a two dimensional array of foci generated with a kinoform.

This paper investigates, through simulation and experiment, the behavior of two dimensional foci arrays generated via phase-only holography where an iterative algorithm was used to produce the kinoforms. Specifically, we studied how aliasing of the signal on a spatial light modulator affects the quality of the foci array as the density and size of the array are varied. This study provides a reference for applications where it is important to understand how the fidelity and overall quality of the foci array changes as the number of foci increases and as the spacing between foci decreases.

Digital in-line X-ray holography with zone plates.

Single pulse imaging with radiation provided by free-electron laser sources is a promising approach towards X-ray microscopy, which is expected to provide high resolution images of biological samples unaffected by radiation damage. One fully coherent imaging technique for this purpose is digital in-line holography. Key to its successful application is the creation of X-ray point sources with high photon flux. In this study we applied zone plates to create such point sources with synchrotron radiation provided by the storage ring BESSY II. The obtained, divergent light cone is applied to holographic microscopy of biological objects such as critical point dried Navicula perminuta diatoms and human cells using photons with an energy of 250 eV. Compared to conventional experiments employing pinholes, exposure times are reduced by two orders of magnitude.

Adding functionalities to precomputed holograms with random mask multiplexing in holographic optical tweezers.

In this study, we present a method designed to generate dynamic holograms in holographic optical tweezers. The approach combines our random mask encoding method with iterative high-efficiency algorithms. This hybrid method can be used to dynamically modify precalculated holograms, giving them new functionalities-temporarily or permanently-with a low computational cost. This allows the easy addition or removal of a single trap or the independent control of groups of traps for manipulating a variety of rigid structures in real time.

Motion of the surface of the human tympanic membrane measured with stroboscopic holography.

Sound-induced motion of the surface of the human tympanic membrane (TM) was studied by stroboscopic holographic interferometery, which measures the amplitude and phase of the displacement at each of about 40,000 points on the surface of the TM. Measurements were made with tonal stimuli of 0.5, 1, 4 and 8 kHz. The magnitude and phase of the sinusoidal displacement of the TM at each driven frequency were derived from the fundamental Fourier component of the raw displacement data computed from stroboscopic holograms of the TM recorded at eight stimulus phases. The correlation between the Fourier estimates and measured motion data was generally above 0.9 over the entire TM surface. We used three data presentations: (i) plots of the phasic displacements along a single chord across the surface of the TM, (ii) phasic surface maps of the displacement of the entire TM surface, and (iii) plots of the Fourier derived amplitude and phase-angle of the surface displacement along four diameter lines that define and bisect each of the four quadrants of the TM. These displays led to some common conclusions: at 0.5 and 1kHz, the entire TM moved roughly in-phase with some small phase delay apparent between local areas of maximal displacement in the posterior half of the TM. At 4 and 8 kHz, the motion of the TM became more complicated with multiple local displacement maxima arranged in rings around the manubrium. The displacements at most of these maxima were roughly in-phase, while some moved out-of-phase. Superposed on this in- and out-of-phase behavior were significant cyclic variations in-phase with location of less than 0.2 cycles or occasionally rapid half-cycle step-like changes in-phase. The high frequency displacement amplitude and phase maps discovered in this study can not be explained by any single wave motion, but are consistent with a combination of low and higher order modal motions plus some small traveling-wave-like components. The observations of the dynamics of TM surface motion from this study will help us better understand the sound-receiving function of the TM and how it couples sound to the ossicular chain and inner ear.