Pregnancy induces retinal microvascular changes indicating cardio-metabolic stress.

We performed longitudinal examinations of the arterial retinal microvasculature using Adaptive Optics Retinal Imaging in a 30-year-old healthy woman with twin pregnancy from the 23rd week of gestation (wog) to three days postpartum. Two blinded graders recorded the average wall-to-lumen ratio (WLR) of the examined retinal artery. There was a significant increase in the mean WLR over the course of pregnancy followed by a decreasing WLR from the 37th wog. The demonstrated changes in WLR may be an expression of vascular remodeling and adaptation to volume load which indicates that pregnancy can be viewed as a cardiovascular stress test.

Assessment of retinal vein pulsation through video-ophthalmoscopy and simultaneous biosignals acquisition.

The phenomenon of retinal vein pulsation is still not a deeply understood topic in retinal hemodynamics. In this paper, we present a novel hardware solution for recording retinal video sequences and physiological signals using synchronized acquisition, we apply the photoplethysmographic principle for the semi-automatic processing of retinal video sequences and we analyse the timing of the vein collapse within the cardiac cycle using of an electrocardiographic signal (ECG). We measured the left eyes of healthy subjects and determined the phases of vein collapse within the cardiac cycle using a principle of photoplethysmography and a semi-automatic image processing approach. We found that the time to vein collapse (Tvc) is between 60 ms and 220 ms after the R-wave of the ECG signal, which corresponds to 6% to 28% of the cardiac cycle. We found no correlation between Tvc and the duration of the cardiac cycle and only a weak correlation between Tvc and age (0.37, p = 0.20), and Tvc and systolic blood pressure (-0.33, p = 0.25). The Tvc values are comparable to those of previously published papers and can contribute to the studies that analyze vein pulsations.

Comparative analysis of retinal photoplethysmographic spatial maps and thickness of retinal nerve fiber layer.

The paper presents a comparative study of the pulsatile attenuation amplitude (PAA) within the optic nerve head (ONH) at four different areas calculated from retinal video sequences and its relevance to the retinal nerve fiber layer thickness (RNFL) changes in normal subjects and patients with different stages of glaucoma. The proposed methodology utilizes processing of retinal video sequences acquired by a novel video ophthalmoscope. The PAA parameter measures the amplitude of heartbeat-modulated light attenuation in retinal tissue. Correlation analysis between PAA and RNFL is performed in vessel-free locations of the peripapillary region with the proposed evaluating patterns: 360° circular area, temporal semi-circle, nasal semi-circle. For comparison, the full ONH area is also included. Various positions and sizes of evaluating patterns in peripapillary region were tested which resulted in different outputs of correlation analysis. The results show significant correlation between PAA and RNFL thickness calculated in proposed areas. The highest correlation coefficient Rtemp = 0.557 (p<0.001) reflects the highest PAA-RNFL correspondence in the temporal semi-circular area, compared to the lowest value in the nasal semi-circular area (Rnasal = 0.332, p<0.001). Furthermore, the results indicate the most relevant approach to calculate PAA from the acquired video sequences is using a thin annulus near the ONH center. Finally, the paper shows the proposed photoplethysmographic principle based on innovative video ophthalmoscope can be used to analyze changes in retinal perfusion in peripapillary area and can be potentially used to assess progression of the RNFL deterioration.

A multi-color video-ophthalmoscopes allows to measure the spectral distribution of light absorption of blood in the human retina.

Based on our previously developed mono-color video-ophthalmoscope a multi-color video-ophthalmoscope was developed. Using narrow band transmission filters, this instrument allows to measure the pulsatile cardiac cycle induced blood volume changes in the human retina for any wavelength in the sensitivity range of the used CMOS-camera. In this key experiment, video sequences (8 s, 25 fps, 200 frames) of the optic nerve head (ONH) were acquire for seven wavelengths between 475 nm and 677 nm one after the other. After image registration of all frames of each video sequence (to compensate for eye movements) and trend correction (to compensate for slow intensity changes), the amplitude of the cardiac cycle induced light intensity changes (pulsatile absorption amplitude PAA) can be calculated for all seven wavelengths. The results confirmed that the spectral distribution of PAA (λ) follows the distribution of the light absorption of blood. The measured values correspond to the absorption of a thin blood layer of about 0.5 µm thickness.

Comprehensive automatic processing and analysis of adaptive optics flood illumination retinal images on healthy subjects.

This work presents a novel fully automated method for retinal analysis in images acquired with a flood illuminated adaptive optics retinal camera (AO-FIO). The proposed processing pipeline consists of several steps: First, we register single AO-FIO images in a montage image capturing a larger retinal area. The registration is performed by combination of phase correlation and the scale-invariant feature transform method. A set of 200 AO-FIO images from 10 healthy subjects (10 images from left eye and 10 images from right eye) is processed into 20 montage images and mutually aligned according to the automatically detected fovea center. As a second step, the photoreceptors in the montage images are detected using a method based on regional maxima localization, where the detector parameters were determined with Bayesian optimization according to manually labeled photoreceptors by three evaluators. The detection assessment, based on Dice coefficient, ranges from 0.72 to 0.8. In the next step, the corresponding density maps are generated for each of the montage images. As a final step, representative averaged photoreceptor density maps are created for the left and right eye and thus enabling comprehensive analysis across the montage images and a straightforward comparison with available histological data and other published studies. Our proposed method and software thus enable us to generate AO-based photoreceptor density maps for all measured locations fully automatically, and thus it is suitable for large studies, as those are in pressing need for automated approaches. In addition, the application MATADOR (MATlab ADaptive Optics Retinal Image Analysis) that implements the described pipeline and the dataset with photoreceptor labels are made publicly available.

Heart rate and age modulate retinal pulsatile patterns.

Theoretical models of retinal hemodynamics showed the modulation of retinal pulsatile patterns (RPPs) by heart rate (HR), yet in-vivo validation and scientific merit of this biological process is lacking. Such evidence is critical for result interpretation, study design, and (patho-)physiological modeling of human biology spanning applications in various medical specialties. In retinal hemodynamic video-recordings, we characterize the morphology of RPPs and assess the impact of modulation by HR or other variables. Principal component analysis isolated two RPPs, i.e., spontaneous venous pulsation (SVP) and optic cup pulsation (OCP). Heart rate modulated SVP and OCP morphology (pFDR < 0.05); age modulated SVP morphology (pFDR < 0.05). In addition, age and HR demonstrated the effect on between-group differences. This knowledge greatly affects future study designs, analyses of between-group differences in RPPs, and biophysical models investigating relationships between RPPs, intracranial, intraocular pressures, and cardiovascular physiology.

Multispectral retinal video-ophthalmoscope with fiber optic illumination.

Multispectral imaging is used in various applications including astronomy, industry and agriculture. In retinal imaging, the single-shot multispectral image stack is typically acquired and analyzed. This multispectral analysis can provide information on various structural or metabolic properties. This paper describes the multispectral improvement of a video-ophthalmoscope, which can acquire retinal video sequences of the optic nerve head and peripapillary area using various spectral light illumination. The description of the multispectral video imaging is provided and several applications are described. These applications include multispectral retinal photoplethysmography, visualization of spontaneous vein pulsation and multispectral RGB image generation.

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction.

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.

Experimental Evaluation of Dry Powder Inhalers during Inhalation and Exhalation Using a Model of the Human Respiratory System (xPULM™).

Dry powder inhalers are used by a large number of patients worldwide to treat respiratory diseases. The objective of this work is to experimentally investigate changes in aerosol particle diameter and particle number concentration of pharmaceutical aerosols generated by four dry powder inhalers under realistic inhalation and exhalation conditions. To simulate patients undergoing inhalation therapy, the active respiratory system model (xPULM™) was used. A mechanical upper airway model was developed, manufactured, and introduced as a part of the xPULM™ to represent the human upper respiratory tract with high fidelity. Integration of optical aerosol spectrometry technique into the setup allowed for evaluation of pharmaceutical aerosols. The results show that there is a significant difference (p < 0.05) in mean particle diameter between inhaled and exhaled particles with the majority of the particles depositing in the lung, while particles with the size of (>0.5 µm) are least influenced by deposition mechanisms. The fraction of exhaled particles ranges from 2.13% (HandiHaler®) over 2.94% (BreezHaler®), and 6.22% (Turbohaler®) to 10.24% (Ellipta®). These values are comparable to previously published studies. Furthermore, the mechanical upper airway model increases the resistance of the overall system and acts as a filter for larger particles (>3 µm). In conclusion, the xPULM™ active respiratory system model is a viable option for studying interactions of pharmaceutical aerosols and the respiratory tract regarding applicable deposition mechanisms. The model strives to support the reduction of animal experimentation in aerosol research and provides an alternative to experiments with human subjects.

Increasing the Image Contrast via Fast Fluorescence Photobleaching.

This paper focuses on the analysis of image sequences acquired during fast photobleaching using a standard wide-field microscope. We show that the photobleaching rate estimated for each pixel is not constant for the whole field of view, but it provides a new spatially variant parametric image related to the cell structure and diffusion of fluorophores. We also provide an alternative way to estimate a pixel-wise photobleaching rate with significantly less computation time than exponential model fitting.Clinical Relevance- This method provides an additional way how fluorescence photobleaching might be used for increasing the image contrast.

Parametric Deconvolution for Cancer Cells Viscoelasticity Measurements from Quantitative Phase Images.

In this contribution, we focused on optimising a dynamic flow-based shear stress system to achieve a reliable platform for cell shear modulus (stiffness) and viscosity assessment using quantitative phase imaging. The estimation of cell viscoelastic properties is influenced by distortion of the shear stress waveform, which is caused by the properties of the flow system components (i.e., syringe, flow chamber and tubing). We observed that these components have a significant influence on the measured cell viscoelastic characteristics. To suppress this effect, we applied a correction method utilizing parametric deconvolution of the flow system's optimized impulse response. Achieved results were compared with the direct fitting of the Kelvin-Voigt viscoelastic model and the basic steady-state model. The results showed that our novel parametric deconvolution approach is more robust and provides a more reliable estimation of viscosity with respect to changes in the syringe's compliance compared to Kelvin-Voigt model.

Self-supervised pretraining for transferable quantitative phase image cell segmentation.

In this paper, a novel U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance from 0.67 to 0.70 of object-wise intersection over union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

Photoplethysmographic analysis of retinal videodata based on the Fourier domain approach.

Assessment of retinal blood flow inside the optic nerve head (ONH) and the peripapillary area is an important task in retinal imaging. For this purpose, an experimental binocular video ophthalmoscope that acquires precisely synchronized video sequences of the optic nerve head and peripapillary area from both eyes has been previously developed. It enables to compare specific characteristics of both eyes and efficiently detect the eye asymmetry. In this paper, we describe a novel methodology for the analysis of acquired video data using a photoplethysmographic approach. We describe and calculate the pulsatile attenuation amplitude (PAA) spatial map, which quantifies the maximum relative change of blood volume during a cardiac cycle using a frequency domain approach. We also describe in detail the origin of PAA maps from the fundamental (the first) and the second harmonic component of the pulsatile signal, and we compare the results obtained by time-based and frequency-based approaches. In several cases, we show the advantages and possibilities of this device and the appropriate image analysis approach - fast measurement and comparison of blood flow characteristics of both eyes at a glance, the robustness of this approach, and the possibility of easy detection of asymmetry.

DeepFoci: Deep learning-based algorithm for fast automatic analysis of DNA double-strand break ionizing radiation-induced foci.

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.

Imaging video plethysmography shows reduced signal amplitude in glaucoma patients in the area of the microvascular tissue of the optic nerve head.

PURPOSE: To measure parameters of the cardiac cycle-induced pulsatile light absorption signal (plethysmography signal) of the optic nerve head (ONH) and to compare parameters between normal subjects and patients with different stages of glaucoma. PATIENTS AND METHODS: A recently developed video ophthalmoscope was used to acquire short video sequences (10 s) of the ONH. After image registration and trend correction, the pulsatile changing light absorption at the ONH tissue (excluding large vessels) was calculated. The changing light absorption depends on the pulsatile changing blood volume. Various parameters, including peak amplitude, steepness, time-to-peak, full width at half maximum (FWHM), and pulse duration, were calculated for averaged individual pulses (heartbeats) of the plethysmography signal. This method was applied to 19 healthy control subjects and 91 subjects with ocular hypertension, as well as different stages of primary open-angle glaucoma (17 subjects with ocular hypertension, 24 with preperimetric glaucoma, and 50 with perimetric glaucoma). RESULTS: Compared to the normal subjects, significant reductions (p < 0.001) in peak amplitude and steepness were observed in the group of perimetric glaucoma patients, but no significant difference was found for time-to-peak, FWHM, and pulse duration. Peak amplitude and steepness showed high correlations with RNFL thickness (p < 0.001). CONCLUSIONS: The presented low-cost video-ophthalmoscope permits measurement of the plethysmographic signal of the ONH tissue and calculation of different blood flow-related parameters. The reduced values of the amplitude and steepness parameters in perimetric glaucoma patients suggest decreased ONH perfusion and blood volume. This outcome is in agreement with results from other studies using OCT angiography and laser speckle flowgraphy, which confirm reduced capillary density in these patients. Registration site: www.clinicaltrials.gov , Trial registration number: NCT00494923.

Blind Source Separation of Retinal Pulsatile Patterns in Optic Nerve Head Video-Recordings.

Dynamic optical imaging of retinal hemodynamics is a rapidly evolving technique in vision and eye-disease research. Video-recording, which may be readily accessible and affordable, captures several distinct functional phenomena such as the spontaneous venous pulsations (SVP) of central vein or local arterial blood supply etc. These phenomena display specific dynamic patterns that have been detected using manual or semi-automated methods. We propose a pioneering concept in retina video-imaging using blind source separation (BSS) serving as an automated localizer of distinct areas with temporally synchronized hemodynamics. The feasibility of BSS techniques (such as spatial principal component analysis and spatial independent component analysis) and K-means based post-processing method were successfully tested on the monocular and binocular video-ophthalmoscopic (VO) recordings of optic nerve head (ONH) in healthy subjects. BSSs automatically detected three spatially distinct reproducible areas, i.e. SVP, optic cup pulsations (OCP) that included areas of larger vessels in the nasal part of ONH, and "other" pulsations (OP). The K-means post-processing reduced a spike noise from the patterns' dynamics while high linear dependence between the non-filtered and post-processed signals was preserved. Although the dynamics of all patterns were heart rate related, the morphology analysis demonstrated significant phase shifts between SVP and OCP, and between SVP and OP. In addition, we detected low frequency oscillations that may represent respiratory-induced effects in time-courses of the VO recordings.

Cell segmentation methods for label-free contrast microscopy: review and comprehensive comparison.

BACKGROUND: Because of its non-destructive nature, label-free imaging is an important strategy for studying biological processes. However, routine microscopic techniques like phase contrast or DIC suffer from shadow-cast artifacts making automatic segmentation challenging. The aim of this study was to compare the segmentation efficacy of published steps of segmentation work-flow (image reconstruction, foreground segmentation, cell detection (seed-point extraction) and cell (instance) segmentation) on a dataset of the same cells from multiple contrast microscopic modalities. RESULTS: We built a collection of routines aimed at image segmentation of viable adherent cells grown on the culture dish acquired by phase contrast, differential interference contrast, Hoffman modulation contrast and quantitative phase imaging, and we performed a comprehensive comparison of available segmentation methods applicable for label-free data. We demonstrated that it is crucial to perform the image reconstruction step, enabling the use of segmentation methods originally not applicable on label-free images. Further we compared foreground segmentation methods (thresholding, feature-extraction, level-set, graph-cut, learning-based), seed-point extraction methods (Laplacian of Gaussians, radial symmetry and distance transform, iterative radial voting, maximally stable extremal region and learning-based) and single cell segmentation methods. We validated suitable set of methods for each microscopy modality and published them online. CONCLUSIONS: We demonstrate that image reconstruction step allows the use of segmentation methods not originally intended for label-free imaging. In addition to the comprehensive comparison of methods, raw and reconstructed annotated data and Matlab codes are provided.

Estimation of Time Delay Between Artery and Vein Pulsation using Experimental Video-Ophthalmoscope.

The fundus observation by ophthalmoscope is a non-invasive approach for diagnosis of various retinal diseases. The vein and artery pulsation are usually clearly apparent on fundus and might be also important for medical practice. Thus our method focuses on these changes and analyzes the time delay between the pulsation signal detected in the vein and the artery region. Data acquired by an experimental video-ophthalmoscope from five subjects with no eye diseases are analyzed. The analysis is based on the selection of artery and vein regions of interest and computation of averaged brightness within these regions for each frame. These extracted signals are filtered, interpolated and the trend is eliminated. Finally, the delays between artery and vein pulsation signals are determined using phase spectra. The measured delays are in the range of 15 to 95 ms, which is comparable with other published results.

Eye movement analysis using a binocular video-ophthalmoscope.

This paper describes an application of binocular video-ophthalmoscope for binocular eye movement analysis during target fixation. We analyze eye movements during monocular and binocular fixation and the changes in eye position for 73 subjects. We show that the standard deviations of differences between eye shifts for vertical and horizontal movements are higher for binocular fixation with respect to monocular fixation. We also present different examples of eye movement visualization.

Blind Source Separation of Different Retinal Pulsatile Patterns from Simultaneous Long-term Binocular Ophthalmoscopic Video-records.

Optical imaging of retinal hemodynamic function is an important part of ophthalmologic research. Development and inventing of imaging devices and data analysis methods are both just in progress. The current study innovatively implements two blind source separation (BSS) techniques (i.e. spatial Principal Component Analysis - sPCA; and spatial Independent Component Analysis - sICA) in application of an automatic detection and segmentation of a distinct Optic Disc (OD) areas with different hemodynamic properties from a simultaneous binocular video-ophthalmoscopic records. Both methods detected 3 different spatial patterns mostly symmetric over both eyes stable and reproducible over investigated participants, i.e. central Spontaneous Vessel Pulsations (SVPs), inner OD intensity pulsations and other OD pulsations. Dynamics of all mentioned patterns has a periodic character with similar main frequency (possibly corresponding to subject-specific heart rate) but shifted phase decreasing patterns' mutual high cross-correlations. The sICA estimates a higher rate of phase shifts than sPCA.