Off-axis digital lensless holographic microscopy based on spatially multiplexed interferometry.

Significance: Digital holographic microscopy (DHM) is a label-free microscopy technique that provides time-resolved quantitative phase imaging (QPI) by measuring the optical path delay of light induced by transparent biological samples. DHM has been utilized for various biomedical applications, such as cancer research and sperm cell assessment, as well as for in vitro drug or toxicity testing. Its lensless version, digital lensless holographic microscopy (DLHM), is an emerging technology that offers size-reduced, lightweight, and cost-effective imaging systems. These features make DLHM applicable, for example, in limited resource laboratories, remote areas, and point-of-care applications. Aim: In addition to the abovementioned advantages, in-line arrangements for DLHM also include the limitation of the twin-image presence, which can restrict accurate QPI. We therefore propose a compact lensless common-path interferometric off-axis approach that is capable of quantitative imaging of fast-moving biological specimens, such as living cells in flow. Approach: We suggest lensless spatially multiplexed interferometric microscopy (LESSMIM) as a lens-free variant of the previously reported spatially multiplexed interferometric microscopy (SMIM) concept. LESSMIM comprises a common-path interferometric architecture that is based on a single diffraction grating to achieve digital off-axis holography. From a series of single-shot off-axis holograms, twin-image free and time-resolved QPI is achieved by commonly used methods for Fourier filtering-based reconstruction, aberration compensation, and numerical propagation. Results: Initially, the LESSMIM concept is experimentally demonstrated by results from a resolution test chart and investigations on temporal stability. Then, the accuracy of QPI and capabilities for imaging of living adherent cell cultures is characterized. Finally, utilizing a microfluidic channel, the cytometry of suspended cells in flow is evaluated. Conclusions: LESSMIM overcomes several limitations of in-line DLHM and provides fast time-resolved QPI in a compact optical arrangement. In summary, LESSMIM represents a promising technique with potential biomedical applications for fast imaging such as in imaging flow cytometry or sperm cell analysis.

Holographic mixed reality for planning transcatheter aortic valve replacement.

BACKGROUND: Using three-dimensional (3D) modalities for optimal pre-procedure planning in transcatheter aortic valve replacement (TAVR) is critical for procedural success. However, current methods rely on visualizing images on a two-dimensional screen, using shading and colors to create the illusion of 3D, potentially impeding the accurate comprehension of the actual anatomy structures. In contrast, a new Mixed Reality (MxR) based software enables accurate 3D visualization, imaging manipulation, and quantification of measurements. AIMS: The study aims to evaluate the feasibility, reproducibility, and accuracy of dimensions of the aortic valve complex as measured with a new holographic MxR software (ARTICOR®, Artiness srl, Milano, Italy) compared to a widely used software for pre-operative sizing and planning (3mensio Medical Imaging BV, Bilthoven, The Netherlands). METHODS: This retrospective, observational, double-center study enrolled 100 patients with severe aortic stenosis who underwent cardiac computed tomography (CCT) before TAVR. The CCT datasets of volumetric aortic valve images were analyzed using 3Mensio and newly introduced MxR-based software. RESULTS: Ninety-eight percent of the CCT datasets were successfully converted into holographic models. A higher level of agreement between the two software systems was observed for linear metrics (short, long, and average diameter). In comparison, agreement was lower for area, perimeter, and annulus-to-coronary ostia distance measurements. Notably, the annulus area, annular perimeter, left ventricular outflow tract (LVOT) area, and LVOT perimeter were significantly and consistently smaller with the MxR-based software compared to the 3Mensio. Excellent interobserver reliability was demonstrated for most measurements, especially for direct linear measurements. CONCLUSIONS: Linear measurements of the aortic valve complex using MxR-based software are reproducible compared to the standard CCT dataset analyzed with 3Mensio. MxR-based software could represent an accurate tool for the pre-procedural planning of TAVR.

Single-beam digital holographic reconstruction: a phase-support enhanced complex wavefront on phase-only function for twin-image elimination.

Significance: In in-line digital holographic microscopy (DHM), twin-image artifacts pose a significant challenge, and reduction or complete elimination is essential for object reconstruction. Aim: To facilitate object reconstruction using a single hologram, significantly reduce inaccuracies, and avoid iterative processing, a digital holographic reconstruction algorithm called phase-support constraint on phase-only function (PCOF) is presented. Approach: In-line DHM simulations and tabletop experiments employing the sliding-window approach are used to compute the arithmetic mean and variance of the phase values in the reconstructed image. A support constraint mask, through variance thresholding, effectively enabled twin-image artifacts. Results: Quantitative evaluations using metrics such as mean squared error, peak signal-to-noise ratio, and mean structural similarity index show PCOF's superior capability in eliminating twin-image artifacts and achieving high-fidelity reconstructions compared with conventional methods such as angular spectrum and iterative phase retrieval methods. Conclusions: PCOF stands as a promising approach to in-line digital holographic reconstruction, offering a robust solution to mitigate twin-image artifacts and enhance the fidelity of reconstructed objects.

Acoustic-holography-patterned primary hepatocytes possess liver functions.

Acoustic holography (AH), a promising approach for cell patterning, emerges as a powerful tool for constructing novel invitro 3D models that mimic organs and cancers features. However, understanding changes in cell function post-AH remains limited. Furthermore, replicating complex physiological and pathological processes solely with cell lines proves challenging. Here, we employed acoustical holographic lattice to assemble primary hepatocytes directly isolated from mice into a cell cluster matrix to construct a liver-shaped tissue sample. For the first time, we evaluated the liver functions of AH-patterned primary hepatocytes. The patterned model exhibited large numbers of self-assembled spheroids and superior multifarious core hepatocyte functions compared to cells in 2D and traditional 3D culture models. AH offers a robust protocol for long-term in vitro culture of primary cells, underscoring its potential for future applications in disease pathogenesis research, drug testing, and organ replacement therapy.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity.

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

Quantum Brain Dynamics and Virtual Reality.

In this paper we propose a control theory of manipulating holograms in Quantum Brain Dynamics (QBD) involving our subjective experiences, i.e. qualia. We begin with the Lagrangian density in QBD and extend our theory to a hierarchical model involving multiple layers covering the neocortex. We adopt reservoir computing approach or morphological computation to manipulate waveforms of holograms involving our subjective experiences. Numerical simulations performed indicate that the convergence to target waveforms of holograms is realized by external electric fields in QBD in a hierarchy. Our theory can be applied to non-invasive neuronal stimulation of the neocortex and adopted to check whether or not our brain adopts the language of holography. In case the protocol in a brain is discovered and the brain adopts the language of holography, our control theory will be applied to develop virtual reality devices by which our subjective experiences provided by the five senses in the form of qualia are manipulated non-invasively. Then, the information content of qualia might be directly transmitted into our brain without passing through sensory organs.

Probing multiplexed basal dendritic computations using two-photon 3D holographic uncaging.

Basal dendrites of layer 5 cortical pyramidal neurons exhibit Na+ and N-methyl-D-aspartate receptor (NMDAR) regenerative spikes and are uniquely poised to influence somatic output. Nevertheless, due to technical limitations, how multibranch basal dendritic integration shapes and enables multiplexed barcoding of synaptic streams remains poorly mapped. Here, we combine 3D two-photon holographic transmitter uncaging, whole-cell dynamic clamp, and biophysical modeling to reveal how synchronously activated synapses (distributed and clustered) across multiple basal dendritic branches are multiplexed under quiescent and in vivo-like conditions. While dendritic regenerative Na+ spikes promote millisecond somatic spike precision, distributed synaptic inputs and NMDAR spikes regulate gain. These concomitantly occurring dendritic nonlinearities enable multiplexed information transfer amid an ongoing noisy background, including under back-propagating voltage resets, by barcoding the axo-somatic spike structure. Our results unveil a multibranch dendritic integration framework in which dendritic nonlinearities are critical for multiplexing different spatial-temporal synaptic input patterns, enabling optimal feature binding.

A prostate seed implantation robot system based on human-computer interactions: Augmented reality and voice control.

The technology of robot-assisted prostate seed implantation has developed rapidly. However, during the process, there are some problems to be solved, such as non-intuitive visualization effects and complicated robot control. To improve the intelligence and visualization of the operation process, a voice control technology of prostate seed implantation robot in augmented reality environment was proposed. Initially, the MRI image of the prostate was denoised and segmented. The three-dimensional model of prostate and its surrounding tissues was reconstructed by surface rendering technology. Combined with holographic application program, the augmented reality system of prostate seed implantation was built. An improved singular value decomposition three-dimensional registration algorithm based on iterative closest point was proposed, and the results of three-dimensional registration experiments verified that the algorithm could effectively improve the three-dimensional registration accuracy. A fusion algorithm based on spectral subtraction and BP neural network was proposed. The experimental results showed that the average delay of the fusion algorithm was 1.314 s, and the overall response time of the integrated system was 1.5 s. The fusion algorithm could effectively improve the reliability of the voice control system, and the integrated system could meet the responsiveness requirements of prostate seed implantation.

Label-Free Three-Dimensional Morphological Characterization of Cell Death Using Holographic Tomography.

This study presents a novel label-free approach for characterizing cell death states, eliminating the need for complex molecular labeling that may yield artificial or ambiguous results due to technical limitations in microscope resolution. The proposed holographic tomography technique offers a label-free avenue for capturing precise three-dimensional (3D) refractive index morphologies of cells and directly analyzing cellular parameters like area, height, volume, and nucleus/cytoplasm ratio within the 3D cellular model. We showcase holographic tomography results illustrating various cell death types and elucidate distinctive refractive index correlations with specific cell morphologies complemented by biochemical assays to verify cell death states. These findings hold promise for advancing in situ single cell state identification and diagnosis applications.

Digital in-line holographic microscopy for label-free identification and tracking of biological cells.

Digital in-line holographic microscopy (DIHM) is a non-invasive, real-time, label-free technique that captures three-dimensional (3D) positional, orientational, and morphological information from digital holographic images of living biological cells. Unlike conventional microscopies, the DIHM technique enables precise measurements of dynamic behaviors exhibited by living cells within a 3D volume. This review outlines the fundamental principles and comprehensive digital image processing procedures employed in DIHM-based cell tracking methods. In addition, recent applications of DIHM technique for label-free identification and digital tracking of various motile biological cells, including human blood cells, spermatozoa, diseased cells, and unicellular microorganisms, are thoroughly examined. Leveraging artificial intelligence has significantly enhanced both the speed and accuracy of digital image processing for cell tracking and identification. The quantitative data on cell morphology and dynamics captured by DIHM can effectively elucidate the underlying mechanisms governing various microbial behaviors and contribute to the accumulation of diagnostic databases and the development of clinical treatments.

Airy-beam holographic sonogenetics for advancing neuromodulation precision and flexibility.

Advancing our understanding of brain function and developing treatments for neurological diseases hinge on the ability to modulate neuronal groups in specific brain areas without invasive techniques. Here, we introduce Airy-beam holographic sonogenetics (AhSonogenetics) as an implant-free, cell type-specific, spatially precise, and flexible neuromodulation approach in freely moving mice. AhSonogenetics utilizes wearable ultrasound devices manufactured using 3D-printed Airy-beam holographic metasurfaces. These devices are designed to manipulate neurons genetically engineered to express ultrasound-sensitive ion channels, enabling precise modulation of specific neuronal populations. By dynamically steering the focus of Airy beams through ultrasound frequency tuning, AhSonogenetics is capable of modulating neuronal populations within specific subregions of the striatum. One notable feature of AhSonogenetics is its ability to flexibly stimulate either the left or right striatum in a single mouse. This flexibility is achieved by simply switching the acoustic metasurface in the wearable ultrasound device, eliminating the need for multiple implants or interventions. AhSonogentocs also integrates seamlessly with in vivo calcium recording via fiber photometry, showcasing its compatibility with optical modalities without cross talk. Moreover, AhSonogenetics can generate double foci for bilateral stimulation and alleviate motor deficits in Parkinson's disease mice. This advancement is significant since many neurological disorders, including Parkinson's disease, involve dysfunction in multiple brain regions. By enabling precise and flexible cell type-specific neuromodulation without invasive procedures, AhSonogenetics provides a powerful tool for investigating intact neural circuits and offers promising interventions for neurological disorders.

Connectomic reconstruction of a female Drosophila ventral nerve cord.

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Insights into polycrystalline microstructure of blood films with 3D Mueller matrix imaging approach.

This study introduces a novel approach in the realm of liquid biopsies, employing a 3D Mueller-matrix (MM) image reconstruction technique to analyze dehydrated blood smear polycrystalline structures. Our research centers on exploiting the unique optical anisotropy properties of blood proteins, which undergo structural alterations at the quaternary and tertiary levels in the early stages of diseases such as cancer. These alterations manifest as distinct patterns in the polycrystalline microstructure of dried blood droplets, offering a minimally invasive yet highly effective method for early disease detection. We utilized a groundbreaking 3D MM mapping technique, integrated with digital holographic reconstruction, to perform a detailed layer-by-layer analysis of partially depolarizing dry blood smears. This method allows us to extract critical optical anisotropy parameters, enabling the differentiation of blood films from healthy individuals and prostate cancer patients. Our technique uniquely combines polarization-holographic and differential MM methodologies to spatially characterize the 3D polycrystalline structures within blood films. A key advancement in our study is the quantitative evaluation of optical anisotropy maps using statistical moments (first to fourth orders) of linear and circular birefringence and dichroism distributions. This analysis provides a comprehensive characterization of the mean, variance, skewness, and kurtosis of these distributions, crucial for identifying significant differences between healthy and cancerous samples. Our findings demonstrate an exceptional accuracy rate of over 90 % for the early diagnosis and staging of cancer, surpassing existing screening methods. This high level of precision and the non-invasive nature of our technique mark a significant advancement in the field of liquid biopsies. It holds immense potential for revolutionizing cancer diagnosis, early detection, patient stratification, and monitoring, thereby greatly enhancing patient care and treatment outcomes. In conclusion, our study contributes a pioneering technique to the liquid biopsy domain, aligning with the ongoing quest for non-invasive, reliable, and efficient diagnostic methods. It opens new avenues for cancer diagnosis and monitoring, representing a substantial leap forward in personalized medicine and oncology.

Delivering clinical tutorials to medical students using the Microsoft HoloLens 2: A mixed-methods evaluation.

BACKGROUND: Mixed reality offers potential educational advantages in the delivery of clinical teaching. Holographic artefacts can be rendered within a shared learning environment using devices such as the Microsoft HoloLens 2. In addition to facilitating remote access to clinical events, mixed reality may provide a means of sharing mental models, including the vertical and horizontal integration of curricular elements at the bedside. This study aimed to evaluate the feasibility of delivering clinical tutorials using the Microsoft HoloLens 2 and the learning efficacy achieved. METHODS: Following receipt of institutional ethical approval, tutorials on preoperative anaesthetic history taking and upper airway examination were facilitated by a tutor who wore the HoloLens device. The tutor interacted face to face with a patient and two-way audio-visual interaction was facilitated using the HoloLens 2 and Microsoft Teams with groups of students who were located in a separate tutorial room. Holographic functions were employed by the tutor. The tutor completed the System Usability Scale, the tutor, technical facilitator, patients, and students provided quantitative and qualitative feedback, and three students participated in semi-structured feedback interviews. Students completed pre- and post-tutorial, and end-of-year examinations on the tutorial topics. RESULTS: Twelve patients and 78 students participated across 12 separate tutorials. Five students did not complete the examinations and were excluded from efficacy calculations. Student feedback contained 90 positive comments, including the technology's ability to broadcast the tutor's point-of-vision, and 62 negative comments, where students noted issues with the audio-visual quality, and concerns that the tutorial was not as beneficial as traditional in-person clinical tutorials. The technology and tutorial structure were viewed favourably by the tutor, facilitator and patients. Significant improvement was observed between students' pre- and post-tutorial MCQ scores (mean 59.2% Vs 84.7%, p < 0.001). CONCLUSIONS: This study demonstrates the feasibility of using the HoloLens 2 to facilitate remote bedside tutorials which incorporate holographic learning artefacts. Students' examination performance supports substantial learning of the tutorial topics. The tutorial structure was agreeable to students, patients and tutor. Our results support the feasibility of offering effective clinical teaching and learning opportunities using the HoloLens 2. However, the technical limitations and costs of the device are significant, and further research is required to assess the effectiveness of this tutorial format against in-person tutorials before wider roll out of this technology can be recommended as a result of this study.

Assessment of pulmonary vascular anatomy: comparing augmented reality by holograms versus standard CT images/reconstructions using surgical findings as reference standard.

BACKGROUND: We compared computed tomography (CT) images and holograms (HG) to assess the number of arteries of the lung lobes undergoing lobectomy and assessed easiness in interpretation by radiologists and thoracic surgeons with both techniques. METHODS: Patients scheduled for lobectomy for lung cancer were prospectively included and underwent CT for staging. A patient-specific three-dimensional model was generated and visualized in an augmented reality setting. One radiologist and one thoracic surgeon evaluated CT images and holograms to count lobar arteries, having as reference standard the number of arteries recorded at surgery. The easiness of vessel identification was graded according to a Likert scale. Wilcoxon signed-rank test and κ statistics were used. RESULTS: Fifty-two patients were prospectively included. The two doctors detected the same number of arteries in 44/52 images (85%) and in 51/52 holograms (98%). The mean difference between the number of artery branches detected by surgery and CT images was 0.31 ± 0.98, whereas it was 0.09 ± 0.37 between surgery and HGs (p = 0.433). In particular, the mean difference in the number of arteries detected in the upper lobes was 0.67 ± 1.08 between surgery and CT images and 0.17 ± 0.46 between surgery and holograms (p = 0.029). Both radiologist and surgeon showed a higher agreement for holograms (κ = 0.99) than for CT (κ = 0.81) and found holograms easier to evaluate than CTs (p < 0.001). CONCLUSIONS: Augmented reality by holograms is an effective tool for preoperative vascular anatomy assessment of lungs, especially when evaluating the upper lobes, more prone to anatomical variations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04227444 RELEVANCE STATEMENT: Preoperative evaluation of the lung lobe arteries through augmented reality may help the thoracic surgeons to carefully plan a lobectomy, thus contributing to optimize patients' outcomes. KEY POINTS: • Preoperative assessment of the lung arteries may help surgical planning. • Lung artery detection by augmented reality was more accurate than that by CT images, particularly for the upper lobes. • The assessment of the lung arterial vessels was easier by using holograms than CT images.

Effect of holographic Guasha on sleep quality of older adults with hypertension in the community: A prospective study.

To explore the effect of holographic Guasha therapy on the Pittsburg Sleep Quality Index (PSQI) and Hamilton Anxiety Rating Scale (HAMA) in older adults with hypertension living in the community. This prospective study was conducted from July 2019 to December 2020. Older adults with hypertension (systolic pressure ≥ 140 mm Hg, diastolic pressure ≥ 90 mm Hg) were divided into the control and Guasha groups. The PSQI and HAMA were assessed before and after 4 weeks of intervention. 62 patients were enrolled, with 31/group (Guasha: 72.4 ±â€…6.9 years, 23.0 ±â€…3.1 kg/m2; control: 71.4 ±â€…6.3 years; 22.9 ±â€…2.9 kg/m2). The total PSQI score did not decrease in the control group after 4 weeks (from 14.8 ±â€…1.2 to 14.8 ±â€…1.8, P = .498) but decreased in the Guasha group (from 14.9 ±â€…1.1 to 6.8 ±â€…3.5, P < .001). All PSQI subscores decreased in the Guasha group after 4 weeks of Guasha intervention (all P < .05), except for the use of sleep medication, since the use of such drugs was an exclusion criterion. The HAMA index scores did not change in the control or Guasha group (both P > .05). Holographic Guasha appears to achieve better sleep outcomes than conventional treatment in improving the sleep quality of older adults with hypertension living in the community. The participants were not randomly assigned to the treatments, and the results should be confirmed in a formal trial.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.

Managing Iatrogenic Aortic Dissection: Insight From 3D Holographic Imaging and CT Computational Fluid Dynamic Simulations.

Iatrogenic aortic dissection is a rare but life-threatening complication of coronary artery bypass surgery. We report a case with incidentally detected iatrogenic aortic dissection related to aorta cross-clamping that was successfully managed with watchful follow-up. The decision making was based on 3-dimensional holographic and fluid dynamic analysis guidance.