Head stabilization apparatus for high-resolution ophthalmic imaging.

Head movement must be stabilized to enable high-quality data collection from optical instrumentation such as eye trackers and ophthalmic imaging devices. Though critically important for imaging, head stabilization is often an afterthought in the design of advanced ophthalmic imaging systems, and experimental devices often adapt used and/or discarded equipment from clinical devices for this purpose. Alternatively, those seeking the most stable solution possible, including many users of adaptive optics ophthalmoscopy systems, utilize bite bars. Bite bars can provide excellent stability but are time consuming to fabricate, decreasing imaging efficiency, and uncomfortable for many patients, especially the elderly and/or those with prosthodontics such as dentures who may refuse participation in a study that requires one. No commercial vendors specifically offer head mount solutions for experimental ophthalmic imaging devices, resulting in nearly every custom device having a different solution for this commonly encountered problem. Parallelizing the head stabilization apparatus across different custom devices may improve standardization of experimental imaging systems for clinical trials and other multicenter investigations. Here we introduce a head mount design for ophthalmic imaging that is modular, adjustable, and customizable to the constraints of different experimental imaging configurations. The three points of head contact in our solution provide excellent stabilization across a range of head sizes and shapes from small children to adults, and the ease of adjustment afforded by our design minimizes the time to get participants stabilized and comfortable.

Long-term satisfaction with curative treatment and follow-up in prostate cancer survivors.

PURPOSE: In a cross-sectional observational study to explore long-term satisfaction with treatment among men who had undergone radical prostatectomy (RP) or definitive pelvic radiotherapy (RT) for prostate cancer (PCa). METHODS: After mean 7 years from therapy (range: 6-8), 431 PCa-survivors (RP: n = 313, RT: n = 118) completed a mailed questionnaire assessing persistent treatment-related adverse effects (AEs) (Expanded Prostate cancer Index Composite [EPIC-26]) and seven Quality indicators describing satisfaction with the health care service following a most often general practitioner (GP)-led follow-up plan. A logistic regression model evaluated the associations between long-term satisfaction and treatment modality, age, the seven satisfaction-related Quality indicators, and persistent AEs. The significance level was set at p< .05. RESULTS: Four of five (81%) PCa-survivors reported long-term satisfaction with their treatment. In a multivariable model, satisfaction was positively associated with sufficient information about treatment and AEs, patient-perceived sufficient cooperation between the hospital and the GP and sufficient follow-up of AEs (ref.: insufficient). Age ≥70 years (ref.: <70) and a rising summary score within the EPIC-26 sexual domain additionally increased long-term satisfaction. The treatment modality itself (RP versus RT) did not significantly impact on satisfaction. CONCLUSIONS: The majority of curatively treated PCa-survivors are satisfied with their treatment more than 5 years after primary therapy. Sufficient information, improved cooperation between the hospital specialists and the responsible GP and optimized follow-up of AEs may further increase long-term satisfaction among prostatectomized and irradiated PCa-survivors.

Simultaneous directional full-field OCT using path-length and carrier multiplexing.

Full-field swept-source optical coherence tomography (FF-SS-OCT) is an emerging technology with potential applications in ophthalmic imaging, microscopy, metrology, and other domains. Here we demonstrate a novel method of multiplexing FF-SS-OCT signals using carrier modulation (CM). The principle of CM could be used to inspect various properties of the scattered light, e.g. its spectrum, polarization, Doppler shift, or distribution in the pupil. The last of these will be explored in this work, where CM was used to acquire images passing through two different optical pupils. The two pupils contained semicircular optical windows with perpendicular orientations, with each window permitting measurement of scattering anisotropy in one dimension by inducing an optical delay between the images formed by the two halves of the pupil. Together, the two forms of multiplexing permit measurement of differential scattering anisotropy in the x and y dimensions simultaneously. To demonstrate the feasibility of this technique our carrier multiplexed directional FF-OCT (CM-D-FF-OCT) system was used to acquire images of a microlens array, human hair, onion skin and in vivo human retina. The results of these studies are presented and briefly discussed in the context of future development and application of this technique.

Optoretinogram: optical measurement of human cone and rod photoreceptor responses to light.

Noninvasive, objective measurement of rod function is as significant as that of cone function, and for retinal diseases such as retinitis pigmentosa and age-related macular degeneration, rod function may be a more sensitive biomarker of disease progression and efficacy of treatment than cone function. Functional imaging of single human rod photoreceptors, however, has proven difficult because their small size and rapid functional response pose challenges for the resolution and speed of the imaging system. Here, we describe light-evoked, functional responses of human rods and cones, measured noninvasively using a synchronized adaptive optics optical coherence tomography (OCT) and scanning light ophthalmoscopy (SLO) system. The higher lateral resolution of the SLO images made it possible to confirm the identity of rods in the corresponding OCT volumes.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

OBJECTIVE: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. DESIGN: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. RESULTS: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. CONCLUSIONS: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.

Acute cerebrovascular event in a dog with polycythemia vera.

A 1-year-old neutered male Labrador retriever mixed breed dog was referred for peracute onset of ataxia and seizures. Hematocrit at presentation was 84%. Magnetic resonance imaging of the brain revealed a lesion in the right caudate nucleus consistent with infarction. Postmortem findings were consistent with polycythemia vera and presumed secondary cerebral infarction.

Événement cérébrovasculaire aigu chez un chien atteint de polycythémie vraie. Un Labrador retriever mâle âgé de 1 an a été référé pour l'apparition suraiguë d'ataxie et de crises d'épilepsie. L'hématocrite était de 84 % à la présentation. L'imagerie par résonance magnétique du cerveau a révélé une lésion dans le noyau caudé droit compatible avec à un infarcissement. Les résultats post mortem étaient conformes à une polycythémie vraie et à un infarcissement cérébral secondaire présumé.(Traduit par Isabelle Vallières).

Parallel line scanning ophthalmoscope for retinal imaging.

A parallel line scanning ophthalmoscope (PLSO) is presented using a digital micromirror device (DMD) for parallel confocal line imaging of the retina. The posterior part of the eye is illuminated using up to seven parallel lines, which were projected at 100 Hz. The DMD offers a high degree of parallelism in illuminating the retina compared to traditional scanning laser ophthalmoscope systems utilizing scanning mirrors. The system operated at the shot-noise limit with a signal-to-noise ratio of 28 for an optical power measured at the cornea of 100 µW. To demonstrate the imaging capabilities of the system, the macula and the optic nerve head of a healthy volunteer were imaged. Confocal images show good contrast and lateral resolution with a 10°×10° field of view.

Ultra-pure platelet isolation from canine whole blood.

BACKGROUND: Several research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs. RESULTS: Using an optimized OptiPrep density gradient technique, platelet recovery was 51.56% with 99.99% platelet purity and leukocyte contamination of 100 leukocytes per 108 platelets, on average. Platelet samples were subjected to additional purification with CD45-labeled Dynabeads after density barrier centrifugation resulting in a 95-fold depletion of residual leukocytes. Platelets purified using these methods remained inactivated as assessed by Annexin V and P-selectin labeling with flow cytometry. CONCLUSIONS: The use of OptiPrep density gradient is a quick method for obtaining highly purified platelet samples from low volumes of canine whole blood with minimal contamination. Additional depletion of residual leukocytes can be achieved using CD45-labeled beads. These platelet samples can then be used for many downstream applications that require ultra-pure platelet samples such as RNA and protein analysis.

Angiography of the retina and the choroid with phase-resolved OCT using interval-optimized backstitched B-scans.

In conventional phase-resolved OCT blood flow is detected from phase changes between successive A-scans. Especially in high-speed OCT systems this results in a short evaluation time interval. This method is therefore often unable to visualize complete vascular networks since low flow velocities cause insufficient phase changes. This problem was solved by comparing B-scans instead of successive A-scans to enlarge the time interval. In this paper a detailed phase-noise analysis of our OCT system is presented in order to calculate the optimal time intervals for visualization of the vasculature of the human retina and choroid. High-resolution images of the vasculature of a healthy volunteer taken with various time intervals are presented to confirm this analysis. The imaging was performed with a backstitched B-scan in which pairs of small repeated B-scans are stitched together to independently control the time interval and the imaged lateral field size. A time interval of ≥ 2.5 ms was found effective to image the retinal vasculature down to the capillary level. The higher flow velocities of the choroid allowed a time interval of 0.64 ms to reveal its dense vasculature. Finally we analyzed depth-resolved histograms of volumetric phase-difference data to assess changes in amount of blood flow with depth. This analysis indicated different flow regimes in the retina and the choroid.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs.

OBJECTIVE: To determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. STUDY DESIGN: Randomized, blocked, crossover design with a 14-day washout period. ANIMALS: Healthy intact female Walker Hounds aged 1-6 years and weighing 20.5-24.2 kg. METHODS: Dogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg(-1), PO, every 12 hours), carprofen (4.4 mg kg(-1), PO, every 24 hours), meloxicam (0.2 mg kg(-1), PO, on the 1st day then 0.1 mg kg(-1), PO, every 24 hours), and deracoxib (2 mg kg(-1), PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B(2) was also measured before and during administration of each drug. RESULTS: All NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg(-1) every 12 hours), carprofen (4.4 mg kg(-1) every 24 hours), meloxicam and deracoxib, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding.

Near infrared autofluorescence imaging of retinal pigmented epithelial cells using 663 nm excitation.

PURPOSE: Fundus autofluorescence (AF) using adaptive optics scanning laser ophthalmoscopy (AOSLO) enables morphometric analysis of individual retinal pigmented epithelial (RPE) cells. However, only a few excitation wavelengths in the visible and near-infrared have been evaluated. Visible light excitation (<600 nm) presents additional safety hazards and is uncomfortable for patients. Near-infrared excitation (>700 nm) overcomes those problems but introduces others, including decreased AF signal and cone signatures that obscure RPE structure. Here we investigated the use of an intermediate wavelength, 663 nm, for excitation and compared it to 795 nm. METHODS: Subjects were imaged using AOSLO equipped with a detection channel to collect AF emission between 814 and 850 nm. Two light sources (663 and 795 nm) were used to excite the retinal fluorophores. We recorded 90 s videos and registered them with custom software to integrate AF images for analysis. RESULTS: We imaged healthy eyes and an eye with pattern dystrophy. Similar AF microstructures were detected with each excitation source, despite ~4 times lower excitation power with 663 nm. The signal-to-noise values showed no meaningful difference between 663 nm and 795 nm excitation and a similar trend was observed for image contrast between the two excitation wavelengths. CONCLUSIONS: Lower light levels can be used with shorter wavelength excitation to achieve comparable images of the microstructure of the RPE as have been obtained using higher light levels at longer wavelengths. Further experiments are needed to fully characterize AF across spectrum and determine the optimal excitation and emission bandwidths that balance efficiency, patient comfort, and efficacy.

Autofluorescent hyperreflective foci on infrared autofluorescence adaptive optics ophthalmoscopy in central serous chorioretinopathy.

Purpose: To test the hypothesis that hyperreflective foci in central serous chorioretinopathy (CSCR) are autofluorescent and may represent macrophages that have engulfed outer retinal fluorophores from the retinal pigment epithelium (RPE) and photoreceptors. Methods: Enrolled subjects underwent spectral domain and swept-source optical coherence tomography, adaptive optics flood-illumination, and adaptive optics scanning laser ophthalmoscopy (AOSLO), including near-infrared autofluorescence (AO-IRAF). For the AO-IRAF imaging, retinal fluorophores were excited using 795 nm light and collected in an emission band from 814 to 850 nm. Results: In 2 of 3 eyes, a hyperautofluorescent signal was detected with an elliptical shape and punctate, granular aspects surrounded by a hypoautofluorescent halo. The size of these structures in the active case was measured to be 17 ± 4 µm in diameter, with at least 45 individual hyperautofluorescent foci identified from the AO-IRAF montage in the active stage of patient 2. In the asymptomatic case there were fewer structures visible (∼10) and their size was smaller (11 ± 4 µm). These hyper-AF foci were colocalized with hyperreflective foci on OCT and visible in simultaneously acquired confocal AOSLO images in active stage. The hyperautofluorescent foci in the patient with active CSCR disappeared coincident with clinical resolution. Conclusion and importance: We show here the first AO-IRAF images from patients with CSCR, demonstrating hyper-autofluorescent punctate foci, colocalized with hyper-reflective foci on confocal AOSLO images and in OCT. The autofluorescence of these foci may be driven by the accumulation of photoreceptor and RPE fluorophores within macrophages during the active stage of the disease.

Design of a radial multi-offset detection pattern for in vivo phase contrast imaging of the inner retina in humans.

Previous work has shown that multi-offset detection in adaptive optics scanning laser ophthalmoscopy (AOSLO) can be used to image transparent cells such as retinal ganglion cells (RGCs) in monkeys and humans. Though imaging in anesthetized monkeys with high light levels produced high contrast images of RGCs, images from humans failed to reach the same contrast due to several drawbacks in the previous dual-wavelength multi-offset approach. Our aim here was to design and build a multi-offset detection pattern for humans at safe light levels that could reveal transparent cells in the retinal ganglion cell layer with a contrast and acquisition time approaching results only previously obtained in monkeys. Here, we present a new single-wavelength solution that allows for increased light power and eliminates problematic chromatic aberrations. Then, we demonstrate that a radial multi-offset detection pattern with an offset distance of 8-10 Airy Disk Diameter (ADD) is optimal to detect photons multiply scattered in all directions from weakly reflective retinal cells thereby enhancing their contrast. This new setup and image processing pipeline led to improved imaging of inner retinal cells, including the first images of microglia with multi-offset imaging in AOSLO.

Multimodal Imaging of Torpedo Maculopathy With Fluorescence Adaptive Optics Imaging of Individual Retinal Pigmented Epithelial Cells.

Torpedo maculopathy (TM) is a rare congenital defect of the retinal pigment epithelium (RPE). The RPE is often evaluated clinically using fundus autofluorescence (AF), a technique that visualizes RPE structure at the tissue level from the intrinsic AF of RPE fluorophores. TM lesions typically emit little or no AF, but this macroscopic assessment is unable to resolve the RPE cells, leaving the organization of the RPE cell mosaic in TM unknown. We used fluorescence adaptive optics scanning laser ophthalmoscopy (AOSLO) to show here for the first time the microscopic cellular-level structural alterations to the RPE cell mosaic in TM that underlie the tissue-level changes seen in conventional clinical imaging. We evaluated two patients with TM using conventional clinical imaging techniques and adaptive optics (AO) infrared autofluorescence (IRAF) in AOSLO. Confocal AOSLO revealed relatively normal cones outside the TM lesion but altered cone appearance within it and along its margins in both patients. We quantified cone topography and RPE cell morphometry from the fovea to the margin of the lesion in case 1 and found cone density to be within the normal range across the locations imaged. However, RPE morphometric analysis revealed disrupted RPE cells outside the margin of the lesion; the mean RPE cell area was greater than two standard deviations above the normative range up to approximately 1.5 mm from the lesion margin. Similar morphometric changes were seen to individual RPE cells in case 2. Multi-modal imaging with AOSLO reveals that RPE cells are abnormal in TM well beyond the margins of the characteristic TM lesion boundary defined with conventional clinical imaging. Since the TM fovea appears to be fully formed, with normal cone packing, it is possible that the congenital RPE defect in TM occurs relatively late in retinal development. This work demonstrates how cellular level imaging of the RPE can provide new insight into RPE pathologies, particularly for rare conditions such as TM.

Kilohertz retinal FF-SS-OCT and flood imaging with hardware-based adaptive optics.

A retinal imaging system was designed for full-field (FF) swept-source (SS) optical coherence tomography (OCT) with cellular resolution. The system incorporates a real-time adaptive optics (AO) subsystem and a very high-speed CMOS sensor, and is capable of acquiring volumetric images of the retina at rates up to 1 kHz. While digital aberration correction (DAC) is an attractive potential alternative to AO, it has not yet been shown to provide resolution allowing visualization of cones in the fovea, where early detection of functional deficits is most critical. Here we demonstrate that FF-SS-OCT with hardware AO permits resolution of foveal cones, imaged at eccentricities of 1° and 2°, with volume rates adequate to measure light-evoked changes in photoreceptors. With the reference arm blocked, the system can operate as a kilohertz AO flood illumination fundus camera with adjustable temporal coherence and is expected to allow measurement of light-evoked changes caused by common path interference in photoreceptor outer segments (OS). In this paper, we describe the system's optical design, characterize its performance, and demonstrate its ability to produce images of the human photoreceptor mosaic.

Microstructure of the retinal pigment epithelium near-infrared autofluorescence in healthy young eyes and in patients with AMD.

Retinal pigmented epithelial (RPE) cells are essential for maintaining normal visual function, especially in their role in the visual cycle, and are thought to be one of the first cell classes affected by age-related macular degeneration (AMD). Clinical imaging systems routinely evaluate the structure of the RPE at the tissue level, but cellular level information may provide valuable RPE biomarkers of health, aging and disease. In this exploratory study, participants were imaged with 795 nm excitation in adaptive optics scanning laser ophthalmoscopy (AOSLO) to observe the microstructure of the near-infrared autofluorescence (AO-IRAF) from the RPE layer in healthy retinas and patients with AMD. The expected hexagonal mosaic of RPE cells was only sometimes seen in normal eyes, while AMD patients exhibited highly variable patterns of altered AO-IRAF. In some participants, AO-IRAF structure corresponding to cones was observed, as we have demonstrated previously. In some AMD patients, marked alterations in the pattern of AO-IRAF could be seen even in areas where the RPE appeared relatively normal in clinical imaging modalities, such as spectral domain optical coherence tomography (SD-OCT). AO-IRAF imaging using AOSLO offers promise for better detection and understanding of early RPE changes in the course of AMD, potentially before clinical signs appear.

Pharmacokinetics of subcutaneous low molecular weight heparin (enoxaparin) in dogs.

Unfractionated heparin has been the standard heparin used in human and veterinary medicine for its anticoagulation effect; however, it has a complex pharmacodynamic profile that requires close monitoring. Low molecular weight heparins have a more predictable bioavailability, allowing standardized dosing without individual patient monitoring. This project was designed to a) evaluate the pharmacokinetics of the subcutaneous (SC) administration of the low molecular weight heparin, enoxaparin, in dogs using anti-Xa activity as a marker of plasma enoxaparin concentrations and b) to establish the dose necessary to maintain activity within an established target range. Enoxaparin at 0.8 mg/kg SC q 6 hours consistently maintained target levels of anti-Xa activity in normal dogs without evidence of hemorrhagic complications.

In vivo retinal imaging for fixational eye motion detection using a high-speed digital micromirror device (DMD)-based ophthalmoscope.

Retinal motion detection with an accuracy of 0.77 arcmin corresponding to 3.7 µm on the retina is demonstrated with a novel digital micromirror device based ophthalmoscope. By generating a confocal image as a reference, eye motion could be measured from consecutively measured subsampled frames. The subsampled frames provide 7.7 millisecond snapshots of the retina without motion artifacts between the image points of the subsampled frame, distributed over the full field of view. An ophthalmoscope pattern projection speed of 130 Hz enabled a motion detection bandwidth of 65 Hz. A model eye with a scanning mirror was built to test the performance of the motion detection algorithm. Furthermore, an in vivo motion trace was obtained from a healthy volunteer. The obtained eye motion trace clearly shows the three main types of fixational eye movements. Lastly, the obtained eye motion trace was used to correct for the eye motion in consecutively obtained subsampled frames to produce an averaged confocal image correct for motion artefacts.

Thromboembolic therapies in dogs and cats: an evidence-based approach.

In veterinary medicine, we are forced to make use of less than ideal "evidence," such as extrapolation from experimental studies in dogs and cats without naturally occurring diseases and from clinical trials in other species (particularly human clinical trials), as well as limited information gained from veterinary clinical experience, small clinical trials, case studies, and anecdotal reports. In this article, specific treatment recommendations are made for each of the common thromboembolic conditions seen in dogs and cats. These recommendations are made with the important caveat that, to date, such suggested therapeutic approaches are based on limited evidence.

Digital micromirror device based ophthalmoscope with concentric circle scanning.

Retinal imaging is demonstrated using a novel scanning light ophthalmoscope based on a digital micromirror device with 810 nm illumination. Concentric circles were used as scan patterns, which facilitated fixation by a human subject for imaging. An annular illumination was implemented in the system to reduce the background caused by corneal reflections and thereby to enhance the signal-to-noise ratio. A 1.9-fold increase in the signal-to-noise ratio was found by using an annular illumination aperture compared to a circular illumination aperture, resulting in a 5-fold increase in imaging speed and a better signal-to-noise ratio compared to our previous system. We tested the imaging performance of our system by performing non-mydriatic imaging on two subjects at a speed of 7 Hz with a maximum 20° (diameter) field of view. The images were shot noise limited and clearly show various anatomical features of the retina with high contrast.