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.

Quantitative Phase Imaging as Sensitive Screening Method for Nanoparticle-Induced Cytotoxicity Assessment.

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.

Linear diattenuation imaging of biological samples with digital lensless holographic microscopy.

A digital lensless holographic microscope (DLHM) sensitive to the linear diattenuation produced by biological samples is reported. The insertion of a linear polarization-states generator and a linear polarization-states analyzer in a typical DLHM setup allows the proper linear diattenuation imaging of microscopic samples. The proposal has been validated for simulated and experimental biological samples containing calcium oxalate crystals extracted from agave leaves and potato starch grains. The performance of the proposed method is similar to that of a traditional polarimetric microscope to obtain linear diattenuation images of microscopic samples but with the advantages of DLHM, such as numerical refocusing, cost effectiveness, and the possibility of field-portable implementation.

Deep Learning-Based Phenotypic Assessment of Red Cell Storage Lesions for Safe Transfusions.

This study presents a novel approach to automatically perform instant phenotypic assessment of red blood cell (RBC) storage lesion in phase images obtained by digital holographic microscopy. The proposed model combines a generative adversarial network (GAN) with marker-controlled watershed segmentation scheme. The GAN model performed RBC segmentations and classifications to develop ageing markers, and the watershed segmentation was used to completely separate overlapping RBCs. Our approach achieved good segmentation and classification accuracy with a Dice's coefficient of 0.94 at a high throughput rate of about 152 cells per second. These results were compared with other deep neural network architectures. Moreover, our image-based deep learning models recognized the morphological changes that occur in RBCs during storage. Our deep learning-based classification results were in good agreement with previous findings on the changes in RBC markers (dominant shapes) affected by storage duration. We believe that our image-based deep learning models can be useful for automated assessment of RBC quality, storage lesions for safe transfusions, and diagnosis of RBC-related diseases.

Open-source, cost-effective, portable, 3D-printed digital lensless holographic microscope.

In this work, the design, construction, and testing of the most cost-effective digital lensless holographic microscope to date are presented. The architecture of digital lensless holographic microscopy (DLHM) is built by means of a 3D-printed setup and utilizing off-the-shelf materials to produce a DLHM microscope costing US$52.82. For the processing of the recorded in-line holograms, an open-source software specifically developed to process this type of recordings is utilized. The presented DLHM setup has all the degrees of freedom needed to achieve different fields of view, levels of spatial resolution, and 2D scanning of the sample. The feasibility of the presented platform is tested by imaging non-bio and bio samples; the resolution test targets, a section of the head of a Drosophila melanogaster fly, red blood cells, and cheek cells are imaged on the built microscope.

Optimizing resource utilization during proficiency-based training of suturing skills in medical students: a randomized controlled trial of faculty-led, peer tutor-led, and holography-augmented methods of teaching.

BACKGROUND: Suturing is a fundamental skill in undergraduate medical education. It can be taught by faculty-led, peer tutor-led, and holography-augmented methods; however, the most educationally effective and cost-efficient method for proficiency-based teaching of suturing is yet to be determined. METHODS: We conducted a randomized controlled trial comparing faculty-led, peer tutor-led, and holography-augmented proficiency-based suturing training in pre-clerkship medical students. Holography-augmented training provided holographic, voice-controlled instructional material. Technical skill was assessed using hand motion analysis every ten sutures and used to construct learning curves. Proficiency was defined by one standard deviation within average faculty surgeon performance. Intervention arms were compared using one-way ANOVA of the number of sutures placed, full-length sutures used, time to proficiency, and incremental costs incurred. Surveys were used to evaluate participant preferences. RESULTS: Forty-four students were randomized to the faculty-led (n = 16), peer tutor-led (n = 14), and holography-augmented (n = 14) intervention arms. At proficiency, there were no differences between groups in the number of sutures placed, full-length sutures used, and time to achieve proficiency. The incremental costs of the holography-augmented method were greater than faculty-led and peer tutor-led instruction ($247.00 ± $12.05, p < 0.001) due to the high cost of the equipment. Faculty-led teaching was the most preferred method (78.0%), while holography-augmented was the least preferred (0%). 90.6% of students reported high confidence in performing simple interrupted sutures, which did not differ between intervention arms (faculty-led 100.0%, peer tutor-led 90.0%, holography-augmented 83.3%, p = 0.409). 93.8% of students felt the program should be offered in the future. CONCLUSION: Faculty-led and peer tutor-led instructional methods of proficiency-based suturing teaching were superior to holography-augmented method with respect to costs and participants' preferences despite being educationally equivalent.

Comparison of 3-Dimensional and Augmented Reality Kidney Models With Conventional Imaging Data in the Preoperative Assessment of Children With Wilms Tumors.

Importance: Nephron-sparing surgery can be considered in well-defined cases of unilateral and bilateral Wilms tumors, but the surgical procedure can be very challenging for the pediatric surgeon to perform. Objective: To assess the added value of personalized 3-dimensional (3-D) kidney models derived from conventional imaging data to enhance preoperative surgical planning. Design, Setting, and Participants: In a survey study, the conventional imaging data of 10 Dutch children with Wilms tumors were converted to 3-D prints and augmented reality (AR) holograms and a panel of pediatric oncology surgeons (n = 7) assessed the quality of the different imaging methods during preoperative evaluation. Kidney models were created with 3-D printing and AR using a mixed reality headset for visualization. Main Outcomes and Measures: Differences in the assessment of 4 anatomical structures (tumor, arteries, veins, and urinary collecting structures) using questionnaires. A Likert scale measured differences between the imaging methods, with scores ranging from 1 (completely disagree) to 5 (completely agree). Results: Of the 10 patients, 7 were girls, and the mean (SD) age was 3.7 (1.7) years. Compared with conventional imaging, the 3-D print and the AR hologram models were evaluated by the surgeons to be superior for all anatomical structures: tumor (median scores for conventional imaging, 4.07; interquartile range [IQR], 3.62-4.15 vs 3-D print, 4.67; IQR, 4.14-4.71; P = .008 and AR hologram, 4.71; IQR, 4.26-4.75; P = .002); arteries (conventional imaging, 3.62; IQR, 3.43-3.93 vs 3-D print, 4.54; IQR, 4.32-4.71; P = .002 and AR hologram, 4.83; IQR, 4.64-4.86; P < .001), veins (conventional imaging, 3.46; IQR 3.39-3.62 vs 3-D print, 4.50; IQR, 4.39-4.68; P < .001 and AR hologram, 4.83; IQR, 4.71-4.86; P < .001), and urinary collecting structures (conventional imaging, 2.76; IQR, 2.42-3.00 vs 3-D print, 3.86; IQR, 3.64-4.39; P < .001 and AR hologram, 4.00; IQR, 3.93-4.58; P < .001). There were no differences in anatomical assessment between the two 3-D techniques (the 3-D print and AR hologram). Conclusions and Relevance: In this study, the 3-D kidney models were associated with improved anatomical understanding among the surgeons and can be helpful in future preoperative planning of nephron-sparing surgery for Wilms tumors. These models may be considered as a supplementary visualization in clinical care.

Combined Raman and polarization sensitive holographic imaging for a multimodal label-free assessment of human sperm function.

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.

Quantitative assessment of cancer cell morphology and motility using telecentric digital holographic microscopy and machine learning.

The noninvasive, fast acquisition of quantitative phase maps using digital holographic microscopy (DHM) allows tracking of rapid cellular motility on transparent substrates. On two-dimensional surfaces in vitro, MDA-MB-231 cancer cells assume several morphologies related to the mode of migration and substrate stiffness, relevant to mechanisms of cancer invasiveness in vivo. The quantitative phase information from DHM may accurately classify adhesive cancer cell subpopulations with clinical relevance. To test this, cells from the invasive breast cancer MDA-MB-231 cell line were cultured on glass, tissue-culture treated polystyrene, and collagen hydrogels, and imaged with DHM followed by epifluorescence microscopy after staining F-actin and nuclei. Trends in cell phase parameters were tracked on the different substrates, during cell division, and during matrix adhesion, relating them to F-actin features. Support vector machine learning algorithms were trained and tested using parameters from holographic phase reconstructions and cell geometric features from conventional phase images, and used to distinguish between elongated and rounded cell morphologies. DHM was able to distinguish between elongated and rounded morphologies of MDA-MB-231 cells with 94% accuracy, compared to 83% accuracy using cell geometric features from conventional brightfield microscopy. This finding indicates the potential of DHM to detect and monitor cancer cell morphologies relevant to cell cycle phase status, substrate adhesion, and motility. © 2017 International Society for Advancement of Cytometry.

Computational sensing of herpes simplex virus using a cost-effective on-chip microscope.

Caused by the herpes simplex virus (HSV), herpes is a viral infection that is one of the most widespread diseases worldwide. Here we present a computational sensing technique for specific detection of HSV using both viral immuno-specificity and the physical size range of the viruses. This label-free approach involves a compact and cost-effective holographic on-chip microscope and a surface-functionalized glass substrate prepared to specifically capture the target viruses. To enhance the optical signatures of individual viruses and increase their signal-to-noise ratio, self-assembled polyethylene glycol based nanolenses are rapidly formed around each virus particle captured on the substrate using a portable interface. Holographic shadows of specifically captured viruses that are surrounded by these self-assembled nanolenses are then reconstructed, and the phase image is used for automated quantification of the size of each particle within our large field-of-view, ~30 mm2. The combination of viral immuno-specificity due to surface functionalization and the physical size measurements enabled by holographic imaging is used to sensitively detect and enumerate HSV particles using our compact and cost-effective platform. This computational sensing technique can find numerous uses in global health related applications in resource-limited environments.

Label-free high temporal resolution assessment of cell proliferation using digital holographic microscopy.

Cell proliferation assays are widely applied in biological sciences to understand the effect of drugs over time. However, current methods often assess cell population growth indirectly, that is, the cells are not actually counted. Instead other parameters, for example, the amount of protein, are determined. These methods often also demand phototoxic labels, have low temporal resolution, or employ end-point assays, and frequently are labor intensive. We have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using digital holographic microscopy (DHM), one of many quantitative phase microscopy techniques. As no labels or stains are required, and only very low intensity illumination is necessary, the technique allows for noninvasive continuous cell counting. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. The developed direct assay was validated by analyzing cell cultures treated with various concentrations of the anti-cancer drug etoposide, a well-established topoisomerase inhibitor that causes DNA damage and leads to programmed cell death. After treatment, the unstained adherent cells were nondestructively imaged every 30 min for 36 h inside a cell incubator. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. Our results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods. © 2017 International Society for Advancement of Cytometry.

Non-invasive, quantitative assessment of the morphology of γ-irradiated human mesenchymal stem cells and periosteal cells using digital holographic microscopy.

PURPOSE: To assure the quality of cells to be used in cell therapy, we examined the applicability of digital holographic microscopy (DHM) for non-invasive, quantitative assessment of changes in cell morphology. MATERIALS AND METHODS: Mesenchymal stem cells derived from adipose tissue (MSC-AT) and bone marrow (MSC-BM), in addition to human alveolar periosteal cells (PC) as a reference, were γ-ray irradiated (1 and 4 Gy), and their morphological changes were quantified without fixation using holographic microscopy. After detachment and fixation with ethanol, cell number and surface antigen expression were determined using an automated cell counter kit and flow-cytometry, respectively. RESULTS: Among various indexes, only indexes related to cell size were significantly changed after γ-irradiation. Both BMC-AT and BMC-BM were enlarged and more sensitive to a low dose of γ-irradiation than PC. In contrast to PC, proteins related to DNA damage repair (γ-H2AX, p21waf1, p53 and Rb) were not substantially upregulated or sustained for a week in either MSC-AT or MSC-BM. CONCLUSION: Instead of DNA damage markers, we suggest that cell morphological parameters (e.g. cell volume) that are monitored by DHM could be a useful and more stable marker of MSC quality.

Holographic Assessment of Lymphoma Tissue (HALT) for Global Oncology Field Applications.

Low-cost, rapid and accurate detection technologies are key requisites to cope with the growing global cancer challenges. The need is particularly pronounced in resource-limited settings where treatment opportunities are often missed due to the absence of timely diagnoses. We herein describe a Holographic Assessment of Lymphoma Tissue (HALT) system that adopts a smartphone as the basis for molecular cancer diagnostics. The system detects malignant lymphoma cells labeled with marker-specific microbeads that produce unique holographic signatures. Importantly, we optimized HALT to detect lymphomas in fine-needle aspirates from superficial lymph nodes, procedures that align with the minimally invasive biopsy needs of resource-constrained regions. We equipped the platform to directly address the practical needs of employing novel technologies for "real world" use. The HALT assay generated readouts in <1.5 h and demonstrated good agreement with standard cytology and surgical pathology.

Holographic assessment of self-phase modulation and blooming in a thermal medium.

The use of a low-power laser beam to characterize self-phase modulation (SPM) and bubble formation during thermal blooming (TB), as well as manipulation of the bubbles, is reported. First, a low-power 633 nm laser beam is used to characterize the induced refractive index profile during SPM of a focused 514 nm pump beam in absorbing liquid media, e.g., a solution of red dye in isopropyl alcohol. The induced phase change is also characterized using digital holography via the 633 nm source as the probe and reference. During TB at higher pump powers, bubble formation occurs in the liquid. Using a modified setup, which minimizes the effects of gravity, buoyancy, and convection, stable bubbles are generated. These are characterized using in-line digital holography with the 633 nm probe beam. It is shown that the bubble size depends on exposure time of the pump and that the bubble can be steered by moving a focused low-power laser beam. Finally, possible applications of these thermally generated bubbles are discussed.

Wide-field computational imaging of pathology slides using lens-free on-chip microscopy.

Optical examination of microscale features in pathology slides is one of the gold standards to diagnose disease. However, the use of conventional light microscopes is partially limited owing to their relatively high cost, bulkiness of lens-based optics, small field of view (FOV), and requirements for lateral scanning and three-dimensional (3D) focus adjustment. We illustrate the performance of a computational lens-free, holographic on-chip microscope that uses the transport-of-intensity equation, multi-height iterative phase retrieval, and rotational field transformations to perform wide-FOV imaging of pathology samples with comparable image quality to a traditional transmission lens-based microscope. The holographically reconstructed image can be digitally focused at any depth within the object FOV (after image capture) without the need for mechanical focus adjustment and is also digitally corrected for artifacts arising from uncontrolled tilting and height variations between the sample and sensor planes. Using this lens-free on-chip microscope, we successfully imaged invasive carcinoma cells within human breast sections, Papanicolaou smears revealing a high-grade squamous intraepithelial lesion, and sickle cell anemia blood smears over a FOV of 20.5 mm(2). The resulting wide-field lens-free images had sufficient image resolution and contrast for clinical evaluation, as demonstrated by a pathologist's blinded diagnosis of breast cancer tissue samples, achieving an overall accuracy of ~99%. By providing high-resolution images of large-area pathology samples with 3D digital focus adjustment, lens-free on-chip microscopy can be useful in resource-limited and point-of-care settings.

Biomedical applications of holographic microspectroscopy [invited].

The identification and quantification of specific molecules are crucial for studying the pathophysiology of cells, tissues, and organs as well as diagnosis and treatment of diseases. Recent advances in holographic microspectroscopy, based on quantitative phase imaging or optical coherence tomography techniques, show promise for label-free noninvasive optical detection and quantification of specific molecules in living cells and tissues (e.g., hemoglobin protein). To provide important insight into the potential employment of holographic spectroscopy techniques in biological research and for related practical applications, we review the principles of holographic microspectroscopy techniques and highlight recent studies.

Calibration of a digital in-line holographic microscopy system: depth of focus and bioprocess analysis.

Digital in-line holographic microscopy (DIHM) allows access to both intensity and phase information with conventional microscopic lateral resolutions. Such imaging techniques can, however, be used to increase the depth of focus compared to conventional compound microscopes. We present a simple DIHM capable of imaging weakly scattering 10 µm diameter microspheres as well as Hs578T cells over a depth of 1 mm; i.e., we demonstrate an increase by a factor of 100 over the depth of focus of a conventional microscope.

Controlled detection in composite nanoresonant array for surface plasmon resonance sensing.

A composite nanoresonant structure is developed for sensitivity enhancement in biorecognition reactions by coupling between the localized resonance and the propagating surface plasmon polariton waves. The resonant structure was accomplished by combining holographic lithography with an oblique metallic deposition for cost-effective, large-area, and reconfigurable fabrication. The metallodielectric nanostructure was assembled with microfluidic channels and examined for biorecognition reactions, which showed pronounced improvement in the limit of detection compared to conventional nanohole array sensing configurations. The temperature influence on the binding affinity and the effectiveness of the control channel were also investigated to demonstrate the capability of the proposed composite nanoresonant surface plasmon resonance array sensor.

Digital holographic microscopy applied to life sciences.

Digital holographic microscopy (DHM) is applied to life sciences applications and demonstrate its capability of real-time imaging and quantitative measurements of physiological parameters such as cell volume or mean cell hemoglobin concentration (MCHC) of erythrocyte cells. DHM has the advantage to be non-invasive (no phototoxicity, no contrast agents) and allows a high throughput measurements.

Virtual reality 3D echocardiography in the assessment of tricuspid valve function after surgical closure of ventricular septal defect.

BACKGROUND: This study was done to investigate the potential additional role of virtual reality, using three-dimensional (3D) echocardiographic holograms, in the postoperative assessment of tricuspid valve function after surgical closure of ventricular septal defect (VSD). METHODS: 12 data sets from intraoperative epicardial echocardiographic studies in 5 operations (patient age at operation 3 weeks to 4 years and bodyweight at operation 3.8 to 17.2 kg) after surgical closure of VSD were included in the study. The data sets were analysed as two-dimensional (2D) images on the screen of the ultrasound system as well as holograms in an I-space virtual reality (VR) system. The 2D images were assessed for tricuspid valve function. In the I-Space, a 6 degrees-of-freedom controller was used to create the necessary projectory positions and cutting planes in the hologram. The holograms were used for additional assessment of tricuspid valve leaflet mobility. RESULTS: All data sets could be used for 2D as well as holographic analysis. In all data sets the area of interest could be identified. The 2D analysis showed no tricuspid valve stenosis or regurgitation. Leaflet mobility was considered normal. In the virtual reality of the I-Space, all data sets allowed to assess the tricuspid leaflet level in a single holographic representation. In 3 holograms the septal leaflet showed restricted mobility that was not appreciated in the 2D echocardiogram. In 4 data sets the posterior leaflet and the tricuspid papillary apparatus were not completely included. CONCLUSION: This report shows that dynamic holographic imaging of intraoperative postoperative echocardiographic data regarding tricuspid valve function after VSD closure is feasible. Holographic analysis allows for additional tricuspid valve leaflet mobility analysis. The large size of the probe, in relation to small size of the patient, may preclude a complete data set. At the moment the requirement of an I-Space VR system limits the applicability in virtual reality 3D echocardiography in clinical practice.