Standard Imaging System Research Articles

Use of Ambient Light Compatible Fluorescence-Guided Surgical Technology for Objective Assessment of Flap Perfusion in Autologous Breast Reconstruction.

Decreased autologous flap vascular perfusion can lead to secondary procedures. Fluorescence angiography during surgery reduces the probability of repeat surgery but suffers from interpretation variability. Recently, the OnLume Avata System was developed, which evaluates real-time vascular perfusion in ambient light. This study aims to predict complications in autologous breast reconstruction using measures of relative intensity (RI) and relative area (RA). Patients undergoing autologous breast reconstruction underwent intraoperative tissue perfusion assessment using the OnLume Avata System. Post-hoc image annotation was completed by labeling areas of the flap interpreted to be "Well Perfused," "Questionably Perfused," and "Under Perfused." RIs and RAs were calculated for the marked areas. Primary complications of interest were overall complication rate, fat and mastectomy skin flap necrosis, and surgical revision. Logistic regression was applied to determine the odds of developing a complication based on RI and RA for each image. A total of 25 patients (45 flaps) were included. In total, 17 patients (68%) developed at least one complication. Patients who developed any complication (p = 0.02) or underwent a surgical revision for complications (p = 0.02) had statistically lower RI of under-perfused portions of the flap. Patients with greater areas of under-perfused flap had a significantly higher risk of developing fat necrosis (odds ratio [OR]: 5.71, p = 0.03) and required a revision operation (OR: 1.10, p = 0.01). Image-based interpretation using the OnLume Avata System correlated with the risk of developing postoperative complications that standard fluorescence imaging systems may not appreciate. This information can benefit surgeons to improve perfusion assessment and intraoperative decision-making.

Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering.

Oxygen concentration measurement in 3D hydrogels is vital in 3D cell culture and tissue engineering. However, standard 3D imaging systems capable of measuring oxygen concentration with adequate precision are based on advanced microscopy platforms, which are not accessible in many laboratories due to the system's complexity and the high price. In this work, we present a fast and low-cost phosphorescence lifetime imaging design for measuring the lifetime of oxygen-quenched phosphorescence emission with 0.25 µs temporal precision and sub-millimeter spatial resolution in 3D. By combining light-sheet illumination and the frequency-domain lifetime measurement using a commercial rolling-shutter CMOS camera in the structure of a conventional optical microscope, this design is highly customizable to accommodate application-specific research needs while also being low-cost as compared to advanced instruments. As a demonstration, we made a fluidic device with a gas-permeable film to create an artificial oxygen gradient in the hydrogel sample. Dye-embedded beads were distributed in the hydrogel to conduct continuous emission lifetime monitoring when nitrogen was pumped through the fluidic channel and changed oxygen distribution in the sample. The dynamics of the changes in lifetime co-related with their location in the gel of size 0.5 mm×1.5 mm×700 µm demonstrate the ability of this design to measure the oxygen concentration stably and precisely in 3D samples.

3D-Printed Microfluidic Perfusion System for Parallel Monitoring of Hydrogel-Embedded Cell Cultures.

The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO2-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications.

Monocular kilometer-scale passive ranging by point-spread function engineering.

Standard imaging systems are designed for 2D representation of objects, while information about the third dimension remains implicit, as imaging-based distance estimation is a difficult challenge. Existing long-range distance estimation technologies mostly rely on active emission of signal, which as a subsystem, constitutes a significant portion of the complexity, size and cost of the active-ranging apparatus. Despite the appeal of alleviating the requirement for signal-emission, passive distance estimation methods are essentially nonexistent for ranges greater than a few hundreds of meters. Here, we present monocular long-range, telescope-based passive ranging, realized by integration of point-spread-function engineering into a telescope, extending the scale of point-spread-function engineering-based ranging to distances where it has never been tested before. We provide experimental demonstrations of the optical system in a variety of challenging imaging scenarios, including adversarial weather conditions, dynamic targets and scenes of diversified textures, at distances extending beyond 1.7 km. We conclude with brief quantification of the effect of atmospheric turbulence on estimation precision, which becomes a significant error source in long-range optical imaging.

Improved exploiting modification direction steganography for hexagonal image processing

Steganography has made significant advances in the Square-pixel-based Image Processing (SIP) domain, but to our knowledge, no work has yet been done in Hexel (Hexagonal Pixel)-based Image Processing (HIP). This paper presents a HIP-domain data hiding method that exploits and improves the SIP-domain Exploiting Modification Direction (EMD) embedding scheme. The proposed method, Hexagonal EMD (HexEMD), utilizes a HIP-domain cover image's hexagonal nature and infrastructure to embed the secret message. In standard digital imaging systems, the sensor portion that converts photonic energy into an analog electrical signal and all the subunits that digitize, process, and display this signal are based on square pixel logic, so there is currently no commercial equipment available to produce HIP-domain images. Thus, the image is first transformed into the HIP domain in software using the infrastructure developed in the project. Then the HIP-domain image is partitioned into non-overlapping heptads of the standard size, each containing seven hexels. Rather than embedding segments to the independent pixel pairs as done in SIP-domain EMD, we do the embedding iteratively in each heptad. Experimental results show that the HexEMD outperforms its SIP equivalent, EMD, by improving embedding capacity and achieving low visual quality distortion.

MEDUSA: An open-source and webcam based multispectral imaging system

Multispectral imaging is at the forefront of contactless surface analysis. Standard multispectral imaging systems use sophisticated software, cameras and light filtering optics. This paper discloses the building of a customizable and cost-effective multispectral imaging and analysis system. It integrates a web camera, light emitting diodes (LEDs) lighting, a semisphere for even lightening, an open-source Arduino™ development board and a free Python application to automatically obtain and visually analyze multispectral images.The device is hereafter called MEDUSA and its optical performance was tested for repeated Imaging consistency, visible and near infrared band sensitivity and lighting evenness. Four proof of concept tests were run in order to understand the advantageous use of this system, as compared to a simple visual score of diverse samples. Each of three qualitative tests used sets of 12 LED band spectral images to analyze ink changes in a counterfeit bill, surface bruises on Hass avocado fruits and transient changes in petri dish grown bacterial colonies. A fourth test used single band imaging in a set of standard laboratory analyzed plant samples, to quantitatively relate a red band light reflectance to its nitrogen content. These tests indicate that MEDUSA made images may yield qualitative and quantitative spectral information unseen to the naked eye, suggesting potential use in currency counterfeit tests, food quality analyses, microbial phenotyping and agricultural plant chemistry.MEDUSA can be freely reproduced and customized from this research, making it a powerful and affordable analytical tool to analyze a wide range of subtle chemical properties in samples at industrial and science fields.

Beeporter: Tools for high-throughput analyses of pollinator-virus infections.

Pollinators are in decline thanks to the combined stresses of disease, pesticides, habitat loss, and climate. Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Understanding host-pathogen interactions and virulence of DWV in honey bees is slowed by the lack of cost-effective high-throughput screening methods for viral infection. Currently, analysis of virus infection in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we describe virus clones tagged with green fluorescent protein (GFP) or nanoluciferase (nLuc) that provide high-throughput detection and quantification of virus infections. GFP fluorescence is measured noninvasively in living bees via commonly available long-wave UV light sources and a smartphone camera, or a standard ultraviolet transilluminator gel imaging system. Nonlethal monitoring with GFP allows continuous screening of virus growth and serves as a direct breeding tool for identifying honey bee parents with increased antiviral resistance. Expression using the nLuc reporter strongly correlates with virus infection levels and is especially sensitive. Using multiple reporters, it is also possible to visualize competition, differential virulence, and host tissue targeting by co-occuring pathogens. Finally, it is possible to directly assess the risk of cross-species "spillover" from honey bees to other pollinators and vice versa.

Reconfigurable Metasurface for Image Processing.

Optical Fourier transform-based processing is an attractive technique due to the fast processing times and large-data rates. Furthermore, it has recently been demonstrated that certain Fourier-based processors can be realized in compact form factors using flat optics. The flat optics, however, have been demonstrated as static filters where the operator is fixed, limiting the applicability of the approach. Here, we demonstrate a reconfigurable metasurface that can be dynamically tuned to provide a range of processing modalities including bright-field imaging, low-pass and high-pass filtering, and second-order differentiation. The dynamically tunable metasurface can be directly combined with standard coherent imaging systems and operates with a numerical aperture up to 0.25 and over a 60 nm bandwidth. The ability to dynamically control light in the wave vector domain, while doing so in a compact form factor, may open new doors to applications in microscopy, machine vision, and sensing.

An Accelerometric Sensor System With Integrated Hydrostatic Pressure Correction to Assess Carotid Arterial Stiffness

Non-invasive evaluation of vascular stiffness has established utility in cardiovascular risk and stroke prediction. This work presents an accelerometric sensor-based system with an integrated hydrostatic pressure correction unit for arterial luminal diameter measurement and local vascular stiffness assessment. A custom-designed accelerometric patch for continuous monitoring of percutaneous acceleration plethysmogram (APG) from the common carotid artery, which could estimate the arterial wall displacement, was developed. A calibration model was established to estimate the true carotid luminal diameter waveform from the accelerometer-based arterial wall displacement signals. The system’s accuracy for carotid stiffness measurement was validated by multiple in-vivo human subject studies and compared against a clinical-grade B-mode ultrasound imaging system. Accelerometric-derived carotid diameter waveform morphologically replicated the anachrotic, and dicrotic limb of the true carotid diameter recorded using reference devices. The group-average baseline value of measured carotid end-diastolic diameter and distension was 5.81 ± 0.53 mm and 0.51 ± 0.15 mm with a maximum observed beat-by-beat variation of 6.5% and 9%, respectively. Accelerometer-based vascular stiffness measures showed a significant correlation (R2 > 0.88, P < 0.0001), and clinically acceptable agreement with a reference standard imaging system. APG derived carotid stiffness indices demonstrated the capability to detect expected age-associated changes in stiffness. The study revealed that the accelerometric sensor system offers a reliable, cost-effective method for long-term non-invasive monitoring of location-specific vascular stiffness measures with potential applications in ambulatory healthcare monitors.

Endoscopic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors in PDT and PDD

Abstract Photodynamic therapy (PDT) is a potential option for treatment of cancer since it can be performed non- invasive for superficial cancers or minimal-invasive with low traumatization. But PDT is intrinsically inefficient due to the complex accumulation of the photosensitizing drug inside the tumor and the processes of heme syntheses to create the needed cell killing components. To optimize the outcome of PDT and increase acceptance as viable option it is necessary to predict the optimal time for the start of the treatment based on measurable precursors. A former cell study proposed a new filter fluorometer in a complex and sensitive setup. In this work we now designed and tested a simplified system that can be used in combination with standard endoscopic imaging systems. This system will be used as base to prove viability of this approach for a future clinical study.

Intravascular optical coherence tomography method for automated detection of macrophage infiltration within atherosclerotic coronary plaques

Background and aimsSignificant macrophages infiltration in advanced atherosclerotic plaques promotes acute coronary events. Hence, the clinical imaging of macrophage content in coronary atherosclerotic plaques could potentially aid in identifying patients most at risk of future acute coronary events. The aim of this study was to introduce and validate a simple intravascular optical coherence tomography (IV-OCT) image processing method for automated, accurate and fast detection of macrophage infiltration within coronary atherosclerotic plaques. MethodsThis method calculates the ratio of the normalized-intensity standard deviation (NSD) values estimated over two axially-adjacent regions of interest in an IV-OCT cross-sectional image (B-scan). When applied to entire IV-OCT B-scans, this method highlights plaque areas with high NSD ratio values (NSDRatio), which was demonstrated to be correlated with the degree of coronary plaque macrophage infiltration. ResultsUsing an optimized NSDRatio threshold value, coronary plaque macrophage infiltration could be detected with ~88% sensitivity and specificity in a database of 28 IV-OCT scans from postmortem coronary segments. For comparison, using an optimized NSD threshold value, considered the standard IV-OCT signature for macrophages, coronary plaque macrophage infiltration could be detected with only ~55% sensitivity and specificity. ConclusionsThe proposed NSDRatio method significantly increases the sensitivity and specificity for the detection of coronary plaque macrophage infiltration compared to the standard NSD method. This computationally efficient method can be seamlessly implemented within standard IV-OCT imaging systems for in-vivo real-time imaging of macrophage content in coronary plaques, which could potentially aid in identifying patients most at risk of future acute coronary events.

Common-path, single-shot phase-shifting digital holographic microscopy using a Ronchi ruling

Phase-shifting digital holography is widely considered to be a groundbreaking method to quantitatively investigate the phase distribution of specimens, such as living cells. The main flaws of this method, however, are that the requirement for several sequential phase-shifted holograms eliminates the possibility of single-shot imaging and complex configurations would also increase the temporal noise. The present paper aims to validate a single-shot, common-path, phase-shifting digital holographic microscopy, employing a self-referencing geometry. A Ronchi ruling, located in the Fourier plane of a standard microscopic imaging system, produces multiple replicas of sample information in the image plane. The phase retrieval algorithm is performed by superposition of the sample-free portion of each replica with the object information, and requires at least three adjacent diffraction orders. To evaluate the performance of the proposed method, the phase distribution of silica microspheres as a test sample and the morphology of red blood cells as a biological specimen are determined. This configuration offers improved temporal stability in comparison with previously reported Mach–Zehnder interferometers, and may serve as an alternative for real-time surveying of nanometric and subnanometric fluctuations of living microscopic specimens.

Scattering Assisted Imaging

Standard imaging systems provide a spatial resolution that is ultimately dictated by the numerical aperture (NA) of the illumination and collection optics. In biological tissues, the resolution is strongly affected by scattering, which limits the penetration depth to a few tenths of microns. Here, we exploit the properties of speckle patterns embedded into a strongly scattering matrix to illuminate the sample at high spatial frequency content. Combining adaptive optics with a custom deconvolution algorithm, we obtain an increase in the transverse spatial resolution by a factor of 2.5 with respect to the natural diffraction limit. Our Scattering Assisted Imaging (SAI) provides an effective solution to increase the resolution when long working distance optics are needed, potentially paving the way to bulk imaging in turbid tissues.

Time-lapse Whole-field Fluorescence Imaging of Microglia ProcessesMotility in Acute Mouse Hippocampal Slices and Analysis.

Microglia are the resident immune cells of the central nervous system (CNS). In the last year, the improvements in the transgenic mouse technologies and imaging techniques have shed light on microglia functions under physiological conditions. Microglia continuously scan the brain parenchyma with their highly motile processes, maintaining tissue homeostasis and participating in neuronal circuits refinement. Here, we describe a protocol that enables us to perform time-lapse imaging of microglial cells in acute hippocampal slices, making image acquisition possible on an electrophysiology rig equipped with a standard imaging system. Using this ex vivo approach, we investigated microglial processes scanning abilities under physiological condition in hippocampus.

Label-Free Electrophoretic Mobility Shift Assay (EMSA) for Measuring Dissociation Constants of Protein-RNA Complexes.

The electrophoretic mobility shift assay (EMSA) is a well-established method to detect formation of complexes between proteins and nucleic acids and to determine, among other parameters, equilibrium constants for the interaction. Mixtures of protein and nucleic acid solutions of various ratios are analyzed via polyacrylamide gel electrophoresis (PAGE) under native conditions. In general, protein-nucleic acid complexes will migrate more slowly than the free nucleic acid. From the distributions of the nucleic acid components in the observed bands in individual gel lanes, quantitative parameters such as the dissociation constant (Kd ) of the interaction can be measured. This article describes a simple and rapid EMSA that relies either on precast commercial or handcast polyacrylamide gels and uses unlabeled protein and nucleic acid. Nucleic acids are instead detected with SYBR Gold stain and band intensities established with a standard gel imaging system. We used this protocol specifically to determine Kd values for complexes between the PAZ domain of Argonaute 2 (Ago2) enzyme and native and chemically modified RNA oligonucleotides. EMSA-based equilibrium constants are compared to those determined with isothermal titration calorimetry (ITC). Advantages and limitations of this simple EMSA are discussed by comparing it to other techniques used for determination of equilibrium constants of protein-RNA interactions, and a troubleshooting guide is provided. © 2018 by John Wiley & Sons, Inc.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular ‘Riboglow’ platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

Image Retrieval in Spatial and Temporal Domains With a Quadrant Detector

To retrieve an N pixel image of a scene, conventional digital cameras record the information using an N pixelated detector with one measurement, while single-pixel imaging achieves it by sampling the image in a particular basis and recording the light intensities of N temporal measurements with a single-pixel detector. Besides these two schemes, it is also possible to obtain the same spatial information with an X pixels detector and N / X measurements, where the pressure of information retrieval is distributed to both spatial and temporal domains, rather than condensed in one domain. In this paper, we investigate this possibility and present a 4-pixels imaging scheme, which retrieves an image partially in spatial domain and partially in temporal domain, by adopting a quadrant detector to a single-pixel imaging system. While yielding images with equal quality, the proof-of-principle system we built is four times faster than a standard single-pixel imaging system, demonstrating the feasibility of the proposed scheme. Our work exploits the flexibility of space-time information retrieval and can be useful for applications where comprise between spatial and temporal domains is needed.

Wide-field color imaging of scatter-based tissue contrast using both high spatial frequency illumination and cross-polarization gating.

This study characterizes the scatter-specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross-polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi-illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide-field imaging. Measurements in tissue-simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter-only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter-based contrast achieved with HSF, CP and HSF-CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter-based contrast within images of tissue. The results suggest that visible CP-HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.

MRI-based electric properties tomography with a quasi-Newton approach

Magnetic resonance electric properties tomography is a non-destructive imaging modality that maps the spatial distribution of the electrical conductivity and permittivity of the human body using standard clinical magnetic resonance imaging systems. From the magnetic field maps and the local form of the Maxwell equations, several schemes have been derived to provide direct approximated formulas but they suffer from instabilities. In this paper, we propose to address it as an inverse problem solved by a constrained optimization algorithm where we exploit the weak formulation of the electric Helmholtz equation and a Lagrangian approach. We derive the associated adjoint field equation and employ a quasi-Newton minimization scheme. We also take advantage of a regularisation strategy based on geometrical a priori information for defining large zones into which the electric parameters are known to be piece-wise constant.

Solid immersion terahertz imaging with sub-wavelength resolution

We have developed a method of solid immersion THz imaging—a non-contact technique employing the THz beam focused into evanescent-field volume and allowing strong reduction in the dimensions of THz caustic. We have combined numerical simulations and experimental studies to demonstrate a sub-wavelength 0.35λ0-resolution of the solid immersion THz imaging system compared to 0.85λ0-resolution of a standard imaging system, employing only an aspherical singlet. We have discussed the prospective of using the developed technique in various branches of THz science and technology, namely, for THz measurements of solid-state materials featuring sub-wavelength variations of physical properties, for highly accurate mapping of healthy and pathological tissues in THz medical diagnosis, for detection of sub-wavelength defects in THz non-destructive sensing, and for enhancement of THz nonlinear effects.