High-Throughput SARS-CoV-2 Antiviral Testing Method Using the Celigo Image Cytometer.

The COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring 2-3 days to complete the initial antiviral assay in biologically relevant cells, and then 3-4 days to visualize and count plaques in Vero cells, or to complete cell extractions and PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. Compared to traditional methods, the assays defined here eliminated on average 3-4 days from our standard processing time for antiviral testing. Moreover, we were able to utilize human cell lines directly that are not typically amenable to PRNT or plaque assays. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.

Segmentation of laser induced retinal lesions using deep learning (December 2021).

OBJECTIVE: Detection of retinal laser lesions is necessary in both the evaluation of the extent of damage from high power laser sources, and in validating treatments involving the placement of laser lesions. However, such lesions are difficult to detect using Color Fundus cameras alone. Deep learning-based segmentation can remedy this, by highlighting potential lesions in the image. METHODS: A unique database of images collected at the Air Force Research Laboratory over the past 30 years was used to train deep learning models for classifying images with lesions and for subsequent segmentation. We investigate whether transferring weights from models that learned classification would improve performance of the segmentation models. We use Pearson's correlation coefficient between the initial and final training phases to reveal how the networks are transferring features. RESULTS: The segmentation models are able to effectively segment a broad range of lesions and imaging conditions. CONCLUSION: Deep learning-based segmentation of lesions can effectively highlight laser lesions, making this a useful tool for aiding clinicians.

Longitudinal neural and vascular structural dynamics produced by chronic microelectrode implantation.

Implanted microelectrode arrays sense local neuronal activity, signals which are used as control commands for brain computer interface (BCI) technology. Patients with tetraplegia have used BCI technology to achieve an extraordinary degree of interaction with their local environment. However, current microelectrode arrays for BCIs lose the ability to record high-quality neural signals in the months-to-years following implantation. Very little is known regarding the dynamic response of neurons and vasculature in the months following electrode array implantation, but loss of structural integrity near the electrode may contribute to the degradation of recording signals. Here, we use in-vivo dual-modality imaging to characterize neuronal and vasculature structures in the same animal for 3 months following electrode insertion. We find ongoing neuronal atrophy, but relative vascular stability, in close proximity to the electrode, along with evidence suggesting links between rare, abrupt hypoxic events and neuronal process atrophy.

Thermal damage thresholds for multiple-pulse porcine skin laser exposures at 1070 nm.

As solid-state laser technology continues to mature, high-energy lasers operating in the near-infrared (NIR) band have seen increased utilization in manufacturing, medical, and military applications. Formulations of maximum permissible exposure limits establish guidelines for the safe use of these systems for a given set of laser parameters, based on past experimental and analytical studies of exposure thresholds causing injury to the skin and eyes. The purpose of our study is to characterize the skin response to multiple-pulsed laser exposures at the NIR wavelength of 1070 nm, at a constant beam diameter of 1 cm, using anesthetized Yucatan mini-pig subjects. Our study explores three constant total laser-on times of 0.01, 0.1, and 10 s as single- and multiple-pulse sequences. Exposures consisting of 10, 30, and 100 pulses have identical individual pulse durations but different duty cycles in order to include variable degrees of thermal additivity. A plurality of three observers quantifies skin damage with the minimally visible lesion metric, judged at the 1- and 24-h intervals postexposure. Calculation of the median effective dose (ED50) provides injury thresholds for all exposure conditions, based on varying laser power across subjects. The results of this study will provide a quantitative basis for the incorporation of multiple-pulsed laser exposure into standards and augment data contained in the existing ED50 database.

Filamentation in Atmospheric Air with Tunable 1100-2400 nm Near-Infrared Femtosecond Laser Source.

Intense femtosecond pulse filamentation in open-air has been utilized for long distance optical communication and remote sensing, but it results in nonlinear-effect driven eye hazards which are not addressed by current eye safety standards. A systematic study of filamentation in atmospheric air was performed using a tunable 100 fs near-infrared laser (1100 nm-2400 nm). While undergoing filamentation, each source wavelength was spectrally broadened resulting in supercontinuum and third harmonic generation in the visible and near-IR spectrum. We record the spectra at the center and fringes of the supercontinuum as it is imaged onto a planar surface. In a full beam collection regime, we report the energy of the sub-1000 nm light generation for source wavelengths from 1100 nm to 1600 nm and compare the energy density to the maximum permissible exposure values under the ANSI Z136.1 laser safety standard.

Acute insertion effects of penetrating cortical microelectrodes imaged with quantitative optical coherence angiography.

The vascular response during cortical microelectrode insertion was measured with amplitude decorrelation-based quantitative optical coherence angiography (OCA). Four different shank-style microelectrode configurations were inserted in murine motor cortex beneath a surgically implanted window in discrete steps while OCA images were collected and processed for angiography and flowmetry. Quantitative measurements included tissue displacement (measured by optical flow), perfused capillary density, and capillary flow velocity. The primary effect of insertion was mechanical perturbation, the effects of which included tissue displacement, arteriolar rupture, and compression of a branch of the anterior cerebral artery causing a global decrease in flow. Other effects observed included local flow drop-out in the region immediately surrounding the microelectrode. The mean basal capillary network velocity for all animals was 0.23 ( ± 0.05 SD ) and 0.18 ( ± 0.07 SD ) mm / s for capillaries from 100 to 300 µ m and 300 to 500 µ m , respectively. Upon insertion, the 2-shank electrode arrays caused a decrease in capillary flow density and velocity, while the results from other configurations were not different from controls. The proximity to large vessels appears to play a larger role than the array configuration. These results can guide neurosurgeons and electrode designers to minimize trauma and ischemia during microelectrode insertion.

Image quality metrics for optical coherence angiography.

We characterized image quality in optical coherence angiography (OCA) en face planes of mouse cortical capillary network in terms of signal-to-noise ratio (SNR) and Weber contrast (Wc) through a novel mask-based segmentation method. The method was used to compare two adjacent B-scan processing algorithms, (1) average absolute difference (AAD) and (2) standard deviation (SD), while varying the number of lateral cross-sections acquired (also known as the gate length, N). AAD and SD are identical at N = 2 and exhibited similar image quality for N<10. However, AAD is relatively less susceptible to bulk tissue motion artifact than SD. SNR and Wc were 15% and 35% higher for AAD from N = 25 to 100. In addition data sets were acquired with two objective lenses with different magnifications to quantify the effect of lateral resolution on fine capillary detection. The lower power objective yielded a significant mean broadening of 17% in Full Width Half Maximum (FWHM) diameter. These results may guide study and device designs for OCA capillary and blood flow quantification.

Nicotine accelerates diabetes-induced retinal changes.

PURPOSE: To investigate the effects of nicotine on retinal alterations in early-stage diabetes in an established rodent model. MATERIALS AND METHODS: Sprague-Dawley rats were examined using a combination of confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography to determine changes in retinal structure in response to nicotine exposure, diabetes and the combined effects of nicotine and diabetes. Diabetes was induced by a single injection of 65 mg/kg streptozotocin and nicotine injections were administered subcutaneously daily. Retinal thickness in the superior, inferior, nasal and temporal quadrants were determined based on the spectral domain optical coherence tomography (SD-OCT) volume scans (20° × 20°) centered on the optic disc. Segmentation of discrete retinal layers was performed on a subset of SD-OCT cross-sections to further examine changes in each treatment group. Survival of neurons within the ganglion cell layer (GCL) was assessed by confocal morphometric imaging. RESULTS: The control group did not experience any significant change throughout the study. The nicotine treatment group experienced an average decrease in total retinal thickness (TRT) of 9.4 µm with the majority of the loss localized within the outer nuclear layer (ONL) as determined by segmentation analysis (p < 0.05). The diabetic group exhibited a trend toward decreased TRT while segmentation analysis of the diabetic retinopathy (DR) group revealed significant thinning within the ONL (p < 0.05). The combination of nicotine and diabetes revealed a significant increase of 8.9 µm in the TRT (p < 0.05) accompanied by a decrease in the number of GCL neurons. CONCLUSIONS: We demonstrated significant temporal changes in retinal morphology in response to nicotine exposure, diabetes and with the combined effects of nicotine and diabetes. These findings may have implications in determining treatment strategies for diabetic patients using products containing nicotine, such as cigarettes, smokeless tobacco, electronic cigarettes or smoking cessation products.

High-resolution multimodal imaging of multiple evanescent white dot syndrome.

The integrity of macular morphology was examined in a patient with multiple evanescent white dot syndrome (MEWDS) diagnosed through clinical and investigational adaptive optics (AO) retinal imaging techniques. Imaging was performed during the acute and recovery phases to examine changes in retinal morphology, revealing characteristic small multifocal white dots in the perifoveal region and a granular appearance in the fovea. Fluorescein angiography revealed early and intermediate hyperfluorescence, and regions of decreased fundus autofluorescence were observed. Photoreceptor disruption was apparent during the acute phase and recurrence. Conventional multimodal imaging combined with AO imaging offers more insight into the pathology of MEWDS by providing complementary views of the retina throughout the acute phase, recovery, and recurrence.

Retinal optical coherence tomography image enhancement via shrinkage denoising using double-density dual-tree complex wavelet transform.

ABSTRACT. Image enhancement of retinal structures, in optical coherence tomography (OCT) scans through denoising, has the potential to aid in the diagnosis of several eye diseases. In this paper, a locally adaptive denoising algorithm using double-density dual-tree complex wavelet transform, a combination of the double-density wavelet transform and the dual-tree complex wavelet transform, is applied to reduce speckle noise in OCT images of the retina. The algorithm overcomes the limitations of commonly used multiple frame averaging technique, namely the limited number of frames that can be recorded due to eye movements, by providing a comparable image quality in significantly less acquisition time equal to an order of magnitude less time compared to the averaging method. In addition, improvements of image quality metrics and 5 dB increase in the signal-to-noise ratio are attained.

Nicotine exposure exacerbates development of cataracts in a type 1 diabetic rat model.

Diabetes and smoking are known risk factors for cataract development. In this study, we evaluated the effect of nicotine on the progression of cataracts in a type 1 diabetic rat model. Diabetes was induced in Sprague-Dawley rats by a single injection of 65 mg/kg streptozotocin. Daily nicotine injections were administered subcutaneously. Forty-five rats were divided into groups of diabetics with and without nicotine treatment and controls with and without nicotine treatment. Progression of lens opacity was monitored using a slit lamp biomicroscope and scores were assigned. To assess whether systemic inflammation played a role in mediating cataractogenesis, we studied serum levels of eotaxin, IL-6, and IL-4. The levels of the measured cytokines increased significantly in nicotine-treated and untreated diabetic animals versus controls and demonstrated a positive trend in the nicotine-treated diabetic rats. Our data suggest the presence of a synergistic relationship between nicotine and diabetes that accelerated cataract formation via inflammatory mediators.

In vivo imaging of photoreceptor disruption associated with age-related macular degeneration: A pilot study.

BACKGROUND AND OBJECTIVE: Age-related macular degeneration is one of the leading causes of vision loss in the developed world. As the disease progresses, the central part of the retina, called the macula, is compromised leading to a disruption of both structure and visual function. In this study, we investigate the disruption of macular photoreceptor cells in vivo as a function of disease stage in patients with the dry form of age-related macular degeneration AMD. MATERIALS AND METHODS: An investigational confocal Adaptive Optics Scanning Laser Ophthalmoscope (AO-SLO) was used to obtain high resolution images of the macular photoreceptor mosaic in patients previously diagnosed with AMD. Four patients were selected as representative cases, comprising each of the four clinical stages of AMD progression. RESULTS: AO-SLO imaging revealed slight disruption in the photoreceptor mosaic in early stage AMD due to focal drusen formation and identified several small drusen deposits that were not observed with standard clinical imaging techniques. An increase in photoreceptor disruption was visualized within the macula in direct correlation with the stage of AMD progression leading to a decrease in visual acuity. Large coalescent drusen and areas of geographic atrophy in advanced stage dry AMD exhibited a significant decrease in visible photoreceptor density. Significant decrease in photoreceptor counts (∼35-50%) were observed when comparing earlier stages of AMD progression (Categories I and II) to later stages of the disease (Categories III and IV). CONCLUSIONS: This study demonstrates the capabilities of adaptive optics retinal imaging to monitor disruption of individual photoreceptor cells as a function of disease progression yielding valuable diagnostic findings in early stage AMD beyond what can be learned about the health of photoreceptors using conventional retinal imaging techniques. Lasers Surg. Med. 44: 603-610, 2012. © 2012 Wiley Periodicals, Inc.

Quantitative evaluation of retinal response to laser photocoagulation using dual-wavelength fundus autofluorescence imaging in a small animal model.

PURPOSE: To demonstrate the usefulness of dual-wavelength fundus autofluorescence (FAF) imaging for noninvasive, quantitative monitoring of dynamic changes associated with healing of retinal photocoagulation lesions in a small animal model. METHODS: Brown Norway rats, exhibiting substantial age-dependent lipofuscin autofluorescence, were used to characterize the kinetics of FAF recovery after retinal photocoagulation. An argon laser with a beam diameter of 100 µm, exposure duration of 0.1 seconds, and a range of laser powers (8-22 mW) were used to create subthreshold, threshold, and suprathreshold lesions. A modified retinal angiograph was used to obtain dual-wavelength FAF images at 488 and 514 nm to quantify and monitor changes in retinal fluorescence up to 6 months. RESULTS: Compared to white light funduscopy, the FAF images exhibited heightened definition and clarity of lesion boundaries immediately after laser exposure. No significant reduction in FAF was measured at or below laser powers of 8 mW. Furthermore, a linear, dose-dependent decrease in FAF (R(2) = 0.9605) was observed immediately after laser exposures of 13 to 22 mW. Complete recovery of baseline FAF was observed for 13.5 and 16 mW exposures at 3 weeks and 4 months, respectively. However, retinal damage was still evident at 6 months after suprathreshold exposure induced using 22 mW laser power. CONCLUSIONS: The accumulation of lipofuscin in the aged Brown Norway rat makes it a suitable small animal model for the characterization of laser-induced injury in the retina based on FAF. Dual-wavelength FAF measurements provide a sensitive, quantitative, noninvasive means of monitoring recovery of laser-induced retinal injury.

Dermal scatter reduction in human skin: a method using controlled application of glycerol.

BACKGROUND AND OBJECTIVE: Previous studies in a hairless Guinea pig model showed that transdermal application of glycerol effected a temporary reduction in dermal scatter of light. This study focuses on the application of this protocol on human patients. STUDY DESIGN/MATERIALS AND METHODS: After stratum corneal removal, glycerol was applied to human subjects using a low pressure transdermal application device. Optical coherence tomography imaging showed increased intensity of radiation reaching deeper regions in the skin and photographs showed enhanced visualization of dermal structures. RESULTS/CONCLUSION: Topically applied glycerol increased light penetration of in vivo corneal-stripped skin. This minimally invasive approach to temporary dermal scatter reduction has the potential to improve the efficacy of light-based diagnostic or therapeutic devices.

Affinity-based turbidity sensor for glucose monitoring by optical coherence tomography: toward the development of an implantable sensor.

We investigated the feasibility of constructing an implantable optical-based sensor for seminoninvasive continuous monitoring of analytes. In this novel sensor, analyte concentration-dependent changes induced in the degree of optical turbidity of the sensing element can be accurately monitored by optical coherence tomography (OCT), an interferometric technique. To demonstrate proof-of-concept, we engineered a sensor for monitoring glucose concentration that enabled us to quantitatively monitor the glucose-specific changes induced in bulk scattering (turbidity) of the sensor. The sensor consists of a glucose-permeable membrane housing that contains a suspension of macroporous hydrogel particles and concanavalin A (ConA), a glucose-specific lectin, that are designed to alter the optical scattering of the sensor as a function of glucose concentration. The mechanism of modulation of bulk turbidity in the sensor is based on glucose-specific affinity binding of ConA to pendant glucose residues of macroporous hydrogel particles. The affinity-based modulation of the scattering coefficient was significantly enhanced by optimizing particle size, particle size distribution, and ConA concentration. Successful operation of the sensor was demonstrated under in vitro condition where excellent reversibility and stability (160 days) of prototype sensors with good overall response over the physiological glucose concentration range (2.5-20 mM) and good accuracy (standard deviation 5%) were observed. Furthermore, to assess the feasibility of using the novel sensor as one that can be implanted under skin, the sensor was covered by a 0.4 mm thick tissue phantom where it was demonstrable that the response of the sensor to 10 mM glucose change could still be measured in the presence of a layer of tissue shielding the sensor aiming to simulate in vivo condition. In summary, we have demonstrated that it is feasible to develop an affinity-based turbidity sensor that can exhibit a highly specific optical response as a function of changes in local glucose concentration and such response can be accurately monitored by OCT suggesting that the novel sensor can potentially be engineered to be used as an implantable sensor for in vivo monitoring of analytes.