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.

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.

[Simulation and Navigation in the Neurosurgical Field Using Three-Dimensional Hholograms by Mixed Reality Devices].

Recently, three-dimensional(3D)holograms from mixed-reality(MR)devices have become available in the medical field. 3D holographic images can provide immersive and intuitive information that has been reported to be very useful for preoperative simulations. Compared with conventional 3D images on a two-dimensional(2D)monitor, 3D holograms offer a higher level of realism, allowing observation of the images anytime and anywhere if the MR device is operational. Even during surgery, surgeons can check realistic 3D holograms in front of them, above the surgical field, without having to turn their heads toward a 2D monitor on the wall. 3D holograms can also be used for neuronavigation if the hologram is tracked to the patient's real head. This method can be defined as 3D augmented reality(AR)navigation, which shows a hologram of a target, such as a tumor or aneurysm, inside the head and brain. In the future, interventions using these techniques with 3D holograms from MR devices are expected to evolve and develop new types of treatments for endoscopic surgery or fluoroscopy-guided endovascular surgery.

Comparative oncology chemosensitivity assay for personalized medicine using low-coherence digital holography of dynamic light scattering from cancer biopsies.

Nearly half of cancer patients who receive standard-of-care treatments fail to respond to their first-line chemotherapy, demonstrating the pressing need for improved methods to select personalized cancer therapies. Low-coherence digital holography has the potential to fill this need by performing dynamic contrast OCT on living cancer biopsies treated ex vivo with anti-cancer therapeutics. Fluctuation spectroscopy of dynamic light scattering under conditions of holographic phase stability captures ultra-low Doppler frequency shifts down to 10 mHz caused by light scattering from intracellular motions. In the comparative preclinical/clinical trials presented here, a two-species (human and canine) and two-cancer (esophageal carcinoma and B-cell lymphoma) analysis of spectral phenotypes identifies a set of drug response characteristics that span species and cancer type. Spatial heterogeneity across a centimeter-scale patient biopsy sample is assessed by measuring multiple millimeter-scale sub-samples. Improved predictive performance is achieved for chemoresistance profiling by identifying red-shifted sub-samples that may indicate impaired metabolism and removing them from the prediction analysis. These results show potential for using biodynamic imaging for personalized selection of cancer therapy.

Label-free cell classification in holographic flow cytometry through an unbiased learning strategy.

Nowadays, label-free imaging flow cytometry at the single-cell level is considered the stepforward lab-on-a-chip technology to address challenges in clinical diagnostics, biology, life sciences and healthcare. In this framework, digital holography in microscopy promises to be a powerful imaging modality thanks to its multi-refocusing and label-free quantitative phase imaging capabilities, along with the encoding of the highest information content within the imaged samples. Moreover, the recent achievements of new data analysis tools for cell classification based on deep/machine learning, combined with holographic imaging, are urging these systems toward the effective implementation of point of care devices. However, the generalization capabilities of learning-based models may be limited from biases caused by data obtained from other holographic imaging settings and/or different processing approaches. In this paper, we propose a combination of a Mask R-CNN to detect the cells, a convolutional auto-encoder, used to the image feature extraction and operating on unlabelled data, thus overcoming the bias due to data coming from different experimental settings, and a feedforward neural network for single cell classification, that operates on the above extracted features. We demonstrate the proposed approach in the challenging classification task related to the identification of drug-resistant endometrial cancer cells.

On-chip label-free cell classification based directly on off-axis holograms and spatial-frequency-invariant deep learning.

We present a rapid label-free imaging flow cytometry and cell classification approach based directly on raw digital holograms. Off-axis holography enables real-time acquisition of cells during rapid flow. However, classification of the cells typically requires reconstruction of their quantitative phase profiles, which is time-consuming. Here, we present a new approach for label-free classification of individual cells based directly on the raw off-axis holographic images, each of which contains the complete complex wavefront (amplitude and quantitative phase profiles) of the cell. To obtain this, we built a convolutional neural network, which is invariant to the spatial frequencies and directions of the interference fringes of the off-axis holograms. We demonstrate the effectiveness of this approach using four types of cancer cells. This approach has the potential to significantly improve both speed and robustness of imaging flow cytometry, enabling real-time label-free classification of individual cells.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Digital holographic technique based breast cancer detection using transfer learning method.

The digital holographic technique is an interferometric method that provides comprehensive information on morphological traits such as cell layer thickness and shape as well as access to biophysical attributes of cells like refractive index, dry mass, and volume. This method helps characterize sample structures in three dimensions both statically and dynamically, even for transparent objects like living biological cells. This research work captures the digital holograms of breast tissues and analyzes the malignancy of the tissue using a deep learning technique. It enables dynamic measurement of the sample under investigation. Different transfer learning models such as Inception, DenseNet, SqueezeNet, VGG, and ResNet are incorporated in this work. The parameters accuracy, precision, sensitivity, and F1 score of different models are compared and found that the ResNet model outperforms better compared to other models.

Testing anti-cancer drugs with holographic incoherent-light-source quantitative phase imaging.

Quantitative Phase Imaging is becoming an important tool in the objective evaluation of cellular responses to experimental treatment. The technique is based on interferometric measurements of the optical thickness of cells in tissue culture reporting on the distribution of dry mass inside the cells. As the measurement of the optical thickness is interferometric, it is not subjected to the Abbe resolution limit, and the use of an incoherent-light source further increases the accuracy practically achieving 0.93nm in optical path difference corresponding to 4.6 femtograms/µm2. Holographic mode reduces the exposure in comparison to phase-shifting or phase-stepping interference microscopy and allows observation of faster dynamics. An attractive application is in the development of novel anti-cancer drugs and there is an important potential for pretesting chemotherapeutic drugs with biopsy material for personalized cancer treatment. The procedure involves the preparation of live cells in tissue culture, seeding them into suitable observation chambers, and time-lapse recording with an adjusted microscope. Subsequent image processing and statistical analysis are essential last steps producing the results, which include rapid measurements of cell growth in terms of dry-mass increase in individual cells, speed of cell motility and other dynamic morphometric parameters.

Holographically Activatable Nanoprobe via Glutathione/Albumin-Mediated Exponential Signal Amplification for High-Contrast Tumor Imaging.

Glutathione (GSH)-activatable probes hold great promise for in vivo cancer imaging, but are restricted by their dependence on non-selective intracellular GSH enrichment and uncontrollable background noise. Here, a holographically activatable nanoprobe caging manganese tetraoxide is shown for tumor-selective contrast enhancement in magnetic resonance imaging (MRI) through cooperative GSH/albumin-mediated cascade signal amplification in tumors and rapid elimination in normal tissues. Once targeting tumors, the endocytosed nanoprobe effectively senses the lysosomal microenvironment to undergo instantaneous decomposition into Mn2+ with threshold GSH concentration of ≈ 0.12 mm for brightening MRI signals, thus achieving high contrast tumor imaging and flexible monitoring of GSH-relevant cisplatin resistance during chemotherapy. Upon efficient up-regulation of extracellular GSH in tumor via exogenous injection, the relaxivity-silent interstitial nanoprobe remarkably evolves into Mn2+ that are further captured/retained and re-activated into ultrahigh-relaxivity-capable complex by stromal albumin in the tumor, and simultaneously allows the renal clearance of off-targeted nanoprobe in the form of Mn2+ via lymphatic vessels for suppressing background noise to distinguish tiny liver metastasis. These findings demonstrate the concept of holographic tumor activation via both tumor GSH/albumin-mediated cascade signal amplification and simultaneous background suppression for precise tumor malignancy detection, surveillance, and surgical guidance.

Discrimination between normal and cancer white blood cells using holographic projection technique.

White blood cells (WBCs) play a vital role in the diagnosis of many blood diseases. Such diagnosis is based on the morphological analysis of blood microscopic images which is performed manually by skilled hematologist. However, this method has many drawbacks, such as the dependence on the hematologist's skill, slow performance, and varying accuracy. Therefore, in the current study, a new optical method for discrimination between normal and cancer WBCs of peripheral blood film (PBF) images is presented. This method is based on holographic projection technique which is able to provide an accurate and fast optical reconstruction method of WBCs floating in the air. Besides, it can provide a 3D visualization map of one WBC with its characterization parameters from only a single 2D hologram. To achieve that, at first, WBCs are accurately segmented from the microscopic PBF images using a developed in-house MATLAB code. Then, their associated phase computer-generated holograms (CGHs) are calculated using the well-known iterative Fourier transform algorithm (IFTA). Within the utilized algorithm, a speckle noise reduction technique, based on temporal multiplexing of spatial frequencies, is applied to minimize the speckle noise across the reconstruction plane. Additionally, a special hologram modulation is added to the calculated holograms to provide a 3D visualization map of one WBC, and discriminate normal and cancer WBCs. Finally, the calculated phase-holograms are uploaded on a phase-only spatial light modulator (SLM) for optical reconstruction. The optical reconstruction of such phase-holograms yields precise representation of normal and cancer WBCs. Moreover, a 3D visualization map of one WBC with its characterization parameters is provided. Therefore, the proposed technique can be used as a valuable tool for interpretation and analysis of WBCs, this in turn could provide an improvement in diagnosis and prognosis of blood diseases.

Novel Dynamic Technique, Nano-DIHM, for Rapid Detection of Oil, Heavy Metals, and Biological Spills in Aquatic Systems.

Numerous anthropogenic and natural particle contaminants exist in diverse aquatic systems, with widely unknown environmental fates. We coupled a flow tube with a digital in-line holographic microscopy (nano-DIHM) technique for aquatic matrices, for in situ real-time analysis of particle size, shape, and phase. Nano-DIHM enables 4D tracking of particles in water and their transformations in three-dimensional space. We demonstrate that nano-DIHM can be automated to detect and track oil spills/oil droplets in dynamic systems. We provide evidence that nano-DIHM can detect the MS2 bacteriophage as a representative biological-viral material and mercury-containing particles alongside other heavy metals as common toxic contaminants. Nano-DIHM shows the capability of observation of combined materials in water, characterizing the interactions of various particles in mixtures, and particles with different coatings in a suspension. The observed sizes of the particles and droplets ranged from ∼1 to 200 µm. We herein demonstrate the ability of nano-DIHM to characterize and distinguish particle-based contaminants in water and their interactions in both stationary and dynamic modes with a 62.5 millisecond time resolution. The fully automated software for dynamic and real-time detection of contaminants will be of global significance. A comparison is also made between nano-DIHM and established techniques such as S/TEM for their different capabilities. Nano-DIHM can provide a range of physicochemical information in stationary and dynamic modes, allowing life cycle analysis of diverse particle contaminants in different aquatic systems, and serve as an effective tool for rapid response for spills and remediation of natural waters.

3D shape reconstruction of normal and cancerous red blood cells using digital holographic tomography: Combination of angular spectrum method and multiplicative technique.

Since the red blood cell shape affects the oxygen transport, so a robust method to reconstruct the 3D shape of an RBC from different projections is presented. A robust one-piece polarizing holographic microscope setup is used to record inline holograms of normal and cancerous red blood cells (RBCs) with high stability. The inline holograms are corrected by flat fielding and windowed Fourier filtering methods to mitigate the zero-order and the defocused twin image due to the inline recording configuration to the least measure. The corrected inline holograms are then reconstructed by the angular spectrum method to extract the 2D wrapping phase-contrast images. The 2D wrapping phase-contrast images are then unwrapped using the graph cuts algorithm to extract the continuous 2D phase-contrast images. The continuous 2D phase-contrast images are reconstructed at different projections by the multiplicative technique to extract the 3D shape of the normal and the cancerous RBCs. Experimental results show that any deformation in the shape of the normal and the cancerous RBCs can be seen clearly at any rotational angle in 3D. This method, which is based on the degree of deformation from the best fitting, can be used as an alternative method of counting method for discrimination between normal and cancerous cells and hence diagnoses the disease easily.

Simple and automatic monitoring of cancer cell invasion into an epithelial monolayer using label-free holographic microscopy.

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner.

Optical Cellular Micromotion: A New Paradigm to Measure Tumor Cells Invasion within Gels Mimicking the 3D Tumor Environments.

Measuring tumor cell invasiveness through 3D tissues, particularly at the single-cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumor invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight into the vast heterogeneity in tumor cell migration through tissues. To address these issues, here the concept of optical cellular micromotion is reported on, where digital holographic microscopy is used to map the optical nano- to submicrometer thickness fluctuations within single-cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. It is experimentally demonstrated that the optical cellular micromotion correlates with tumor cells motility and invasiveness both at the population and single-cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumor cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behavior of single tumor cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumor cell invasiveness.

Application of holography and augmented reality based technology to visualize the internal structure of the dental root - a proof of concept.

BACKGROUND: The Augmented Reality (AR) blends digital information with the real world. Thanks to cameras, sensors, and displays it can supplement the physical world with holographic images. Nowadays, the applications of AR range from navigated surgery to vehicle navigation. DEVELOPMENT: The purpose of this feasibility study was to develop an AR holographic system implementing Vertucci's classification of dental root morphology to facilitate the study of tooth anatomy. It was tailored to run on the AR HoloLens 2 (Microsoft) glasses. The 3D tooth models were created in Autodesk Maya and exported to Unity software. The holograms of dental roots can be projected in a natural setting of the dental office. The application allowed to display 3D objects in such a way that they could be rotated, zoomed in/out, and penetrated. The advantage of the proposed approach was that students could learn a 3D internal anatomy of the teeth without environmental visual restrictions. CONCLUSIONS: It is feasible to visualize internal dental root anatomy with AR holographic system. AR holograms seem to be attractive adjunct for learning of root anatomy.

Characterization of exhaled e-cigarette aerosols in a vape shop using a field-portable holographic on-chip microscope.

The past decade marked a drastic increase in the usage of electronic cigarettes. The adverse health impact of secondhand exposure due to exhaled e-cig particles has raised significant concerns, demanding further research on the characteristics of these particles. In this work, we report direct volatility measurements on exhaled e-cig aerosols using a field-portable device (termed c-Air) enabled by deep learning and lens-free holographic microscopy; for this analysis, we performed a series of field experiments in a vape shop where customers used/vaped their e-cig products. During four days of experiments, we periodically sampled the indoor air with intervals of ~ 16 min and collected the exhaled particles with c-Air. Time-lapse inline holograms of the collected particles were recorded by c-Air and reconstructed using a convolutional neural network yielding phase-recovered microscopic images of the particles. Volumetric decay of individual particles due to evaporation was used as an indicator of the volatility of each aerosol. Volatility dynamics quantified through c-Air experiments showed that indoor vaping increased the percentage of volatile and semi-volatile particles in air. The reported methodology and findings can guide further studies on volatility characterization of indoor e-cig emissions.

Method to analyze effects of low-level laser therapy on biological cells with a digital holographic microscope.

Low-level laser therapy (LLLT) is a therapeutic tool that uses the photobiochemical interaction between light and tissue. Its effectiveness is controversial due to a strong dependence on dosimetric parameters. In this work, we demonstrate that digital holographic microscopy is an effective label-free imaging technique to analyze the effects of LLLT on biological cells, and we propose the full methodology to create correct synthetic aperture phase maps for further extensive, highly accurate statistical analysis. The proposed methodology has been designed to provide a basis for many other biological experiments using quantitative phase imaging. We use SHSY-5Y and HaCaT cells irradiated with different doses of red light for the experiment. The analysis shows quantitative changes in cell dry mass density and the projected cell surface in response to different radiation doses.

Engineered fluidic device to achieve multiplexed monitoring of cell cultures with digital holographic microscopy.

We present a low-cost, 3D-printed, and biocompatible fluidic device, engineered to produce laminar and homogeneous flow over a large field-of-view. Such a fluidic device allows us to perform multiplexed temporal monitoring of cell cultures compatible with the use of various pharmacological protocols. Therefore, specific properties of each of the observed cell cultures can be discriminated simultaneously during the same experiment. This was illustrated by monitoring the agonists-mediated cellular responses, with digital holographic microscopy, of four different cell culture models of cystic fibrosis. Quantitatively speaking, this multiplexed approach provides a time saving factor of around four to reveal specific cellular features.

Digital holographic microscopy: a noninvasive method to analyze the formation of spheroids.

Digital holographic (DH) microscopy is a unique noninvasive method to analyze living cells. With DH microscopy, in vitro cell cultures can be imaged in 2D and pseudo-3D and measurements of size and morphology of the cells are provided. Here, a description of a novel methodology utilizing DH microscopy for the analysis of spheroids is presented. A cell culture protocol is introduced and morphological parameters of cell spheroids as measured by DH microscopy are presented. The study confirms the use of DH microscopy for the analysis of cell spheroids. In the future, organoids can be analyzed with DH microscopy, and it can also be used for drug response and cell death analyses.