Autologous lamellar keratoplasty for the treatment of feline corneal sequestrum: A retrospective study of 35 eyes (2012-2020).

OBJECTIVE: To evaluate the use of autologous lamellar keratoplasty for the treatment of feline corneal sequestrum (FCS). PROCEDURE: The medical records of cats diagnosed with FCS that underwent autologous lamellar keratoplasty between 2012 and 2020 with a minimum of 2 months of follow-up were reviewed. After keratectomy of FCS, a button adjacent to the corneal limbus was harvested on the same eye and sutured to the recipient bed. A nictitating membrane flap was left in place until the first recheck except for one patient. Postoperative treatment with topical and systemic antibiotics and systemic nonsteroidal anti-inflammatory medications was prescribed. Follow-up examinations were carried out 2 weeks, 1 month and 2 months post-operatively and consisted of a complete ophthalmic examination. RESULTS: A total of 35 cats (35 eyes) were included. The median follow-up time was 3.2 months (range, 2-59 months). Brachycephalic cats were overrepresented (85.7%). The mean graft size was 6.5 mm (range, 6-9 mm). Minor complications consisting of melting and partial integration of the graft occurred in 2/35 eyes (5.7%). Recurrence was observed in 1/35 eyes (2.9%) and was managed by a superficial keratectomy. A good visual outcome was achieved in all eyes, and a faint or mild corneal opacification occurred in 15/35 (42.9%). CONCLUSIONS: Autologous lamellar keratoplasty is an effective treatment for FCS, providing good tectonic support to the affected cornea and resulting in good visual and cosmetic outcomes. These results should be verified in future prospective studies that include a larger number of cases and longer-term follow-up.

Zebrafish (Danio rerio) as a model to visualize infection dynamics of Vibrio anguillarum following intraperitoneal injection and bath exposure.

Vaccine development is important for sustainable fish farming and novel vaccines need to be efficacy tested before release to the market. Challenge of fish with the pathogen towards which the vaccine has been produced can be conducted either by external exposure though bathing or cohabitation, or by bypassing the mucosa through injection. The latter approach is often preferred since it is easier to control than the former. However, injection is not a very natural route of infection, and the bypass of the mucosa may result in a different efficacy profile of experimental fish compared to farmed fish, for which the vaccines are targeted. The zebrafish is by now a well established practical vertebrate model species due in part to its size and ease of maintenance and genetic manipulation. Here we use zebrafish as a model to visualize and compare the development of infection of Vibrio anguillarum on and in the fish following injection or bathing. Injection of 103 bacteria per fish resulted in approximately 50% mortality by day 4 post-injection. Similar mortality levels were reached in the other group by bathing in 1.25 × 109 bacteria for 1 min. The spreading of bacteria was followed for the first 24 h after injection/bathing by immunohistochemistry and optical projection tomography. The tissues and organs where bacteria were detected differed significantly as a result of time as well as treatment. In the bath group, bacteria were initially found on external surfaces including gut. After 24 h V. anguillarum still persisted in gut but had now also spread to the blood. In the injection group bacteria were found in the blood throughout all sampling times, as well as in the hypodermis and body cavity at most sampling times.

Optical Projection Tomography as a Novel Method to Visualize and Quantitate Whole-Brain Patterns of Cell Proliferation in the Adult Zebrafish Brain.

How distinct cell populations are distributed in three-dimensional space under homeostasis or following injury, neurodegeneration, or with senescence can teach us much about brain-wide patterns and signaling along the neuroaxis. Visualizing individual cell populations in the mature vertebrate central nervous system (CNS) has remained a challenge as a result of difficulty clearing adult brain tissue or limitations in imaging depth or resolution. We have developed a simple clearing and imaging pipeline optimally suited for the adult zebrafish brain to investigate changes in patterns of cell proliferation in wild-type and transgenic backgrounds that can easily be quantified and represented using FIJI and IMARIS software.

Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects.

Optoacoustic tomography (OT) is now widely used in preclinical imaging; however, the precision (repeatability and reproducibility) of OT has yet to be determined. Methods: We used a commercial small-animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, and frame averaging. Variables due to animal handling and physiology, such as anatomic placement and anesthesia conditions, were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and in vivo. Sensitivity to small-molecule dyes was determined in phantoms and in vivo; precision was assessed in vivo using IRDye800CW. Results: OT COV in a stable phantom was less than 2.8% across all wavelengths over 30 d. The factors with the greatest impact on signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4% at 700 nm. Anatomic region-of-interest size showed the highest variation, at 12% and 18% COV in the kidney and spleen, respectively; however, functional SO2 measurements based on a standard operating procedure showed an exceptional reproducibility of less than 4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for in vivo data included respiration rate, degree of user experience, and animal placement. Conclusion: Data acquired with our small-animal OT system were highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.

Accelerated Optical Projection Tomography Applied to In Vivo Imaging of Zebrafish.

Optical projection tomography (OPT) provides a non-invasive 3-D imaging modality that can be applied to longitudinal studies of live disease models, including in zebrafish. Current limitations include the requirement of a minimum number of angular projections for reconstruction of reasonable OPT images using filtered back projection (FBP), which is typically several hundred, leading to acquisition times of several minutes. It is highly desirable to decrease the number of required angular projections to decrease both the total acquisition time and the light dose to the sample. This is particularly important to enable longitudinal studies, which involve measurements of the same fish at different time points. In this work, we demonstrate that the use of an iterative algorithm to reconstruct sparsely sampled OPT data sets can provide useful 3-D images with 50 or fewer projections, thereby significantly decreasing the minimum acquisition time and light dose while maintaining image quality. A transgenic zebrafish embryo with fluorescent labelling of the vasculature was imaged to acquire densely sampled (800 projections) and under-sampled data sets of transmitted and fluorescence projection images. The under-sampled OPT data sets were reconstructed using an iterative total variation-based image reconstruction algorithm and compared against FBP reconstructions of the densely sampled data sets. To illustrate the potential for quantitative analysis following rapid OPT data acquisition, a Hessian-based method was applied to automatically segment the reconstructed images to select the vasculature network. Results showed that 3-D images of the zebrafish embryo and its vasculature of sufficient visual quality for quantitative analysis can be reconstructed using the iterative algorithm from only 32 projections-achieving up to 28 times improvement in imaging speed and leading to total acquisition times of a few seconds.

Aspectos da tomografia de coerência óptica em cães com retinopatia / Aspects of optical coherence tomography in retinopathy in dogs

A tomografia de coerência óptica (OCT) é um exame não invasivo e de não contato que permite avaliar a retina e o nervo óptico. As imagens da OCT fornecem informações da constituição da retina e sua integridade estrutural in vivo, gerando imagens de alta resolução, que se assemelham à microscopia óptica. Objetivou-se descrever a técnica de tomografia de coerência óptica (OCT) e sua utilização em cães. Foi possível diferenciar claramente as camadas retinianas de cães hígidos e compará-las com as de cães portadores de atrofia progressiva de retina, que apresentaram perda da estratificação e diminuição significativa das camadas. No descolamento de retina (DR) foi possível observar a separação entre a retina neurossensorial e o epitélio pigmentário da retina (EPR), além da presença de exsudatos intrarretinianos. Assim, a OCT mostrou-se eficaz no diagnóstico de retinopatias.

The OCT is a noninvasive and noncontact exam capable to evaluate the retina and optic nerve. The OCT images provide information of the constitution of the retina and its structural integrity in vivo, providing high-resolution images that resemble optical microscopy. The objective of this paper was to describe and document the use of the optical coherence tomography (OCT) in dogs. It was possible differentiate the retinal layers of healthy dogs and compare them with dogs with progressive retinal atrophy which showed altered stratification and significant reduce of the layers. In cases of retinal detachment was observed separation of neurosensory retina from the retinal pigment epithelium, and the presence of intrarretinal exudates. Thus, the OCT was effective in the diagnosis of retinopathy.

Techniques in electron microscopy of animal tissue.

Technical improvements in electron microscopy, both instrumental and preparative, permit increasingly accurate analyses. Digital images for transmission electron microscopy (TEM) can be processed by software programs that automate tasks and create custom tools that allow for image enhancement for brightness, contrast and coloration; for creation of rectangular, ellipsoidal or irregular area selections; and for measurement of mean area and standard deviation. Sample preparation remains a source of error since organelles and spatial arrangements of macromolecules rapidly change after anoxia. Guidelines for maintaining consistency in preparation, examination and interpretation are presented for different electron microscopy (EM) modalities.

Hybrid small animal imaging system combining magnetic resonance imaging with fluorescence tomography using single photon avalanche diode detectors.

The high sensitivity of fluorescence imaging enables the detection of molecular processes in living organisms. However, diffuse light propagation in tissue prevents accurate recovery of tomographic information on fluorophore distribution for structures embedded deeper than 0.5 mm. Combining optical with magnetic resonance imaging (MRI) provides an accurate anatomical reference for fluorescence imaging data and thereby enables the correlation of molecular with high quality structural/functional information. We describe an integrated system for small animal imaging incorporating a noncontact fluorescence molecular tomography (FMT) system into an MRI detector. By adopting a free laser beam design geometrical constraints imposed by the use of optical fibers could be avoided allowing for flexible fluorescence excitation schemes. Photon detection based on a single-photon avalanche diode array enabled simultaneous FMT/MRI measurements without interference between modalities. In vitro characterization revealed good spatial accuracy of FMT data and accurate quantification of dye concentrations. Feasibility of FMT/MRI was demonstrated in vivo by simultaneous assessment of protease activity and tumor morphology in murine colon cancer xenografts.

Combined system of fluorescence diffuse optical tomography and microcomputed tomography for small animal imaging.

We developed a dual-modality system that combines fluorescence diffuse optical tomography (fDOT) and flat panel detector-based microcomputed tomography (micro-CT) to simultaneously reveal molecular and structural information in small animals. In fDOT, a 748 nm diode laser was used as an excitation source, while a cooled charge coupled device camera was adopted to collect transmission fluorescence. In micro-CT, a flat panel detector based on amorphous silicon, with active area of 13 x 13 cm(2), and a microfocus x-ray tube were used. The fDOT system was mounted orthogonally to the micro-CT and the projection images were acquired without rotation of the sample, which is different from the method used for micro-CT alone. Both the finite element method and the algebraic reconstruction technique were used to reconstruct images from the fDOT. Phantom data showed that the resolution of the fDOT system was about 3 mm at an imaging depth of 7 mm. Quantitative error was no more than 5% and imaging sensitivity for 1,1(')-dioctadecyl-3,3,3('),3(')-etramethylindotricarbocyanine iodide bis-oleate (DiR-BOA) was estimated to be higher than 100 nM at a depth of 7 mm. Calculations of the phantom's center of mass showed that the location accuracy of fDOT was about 0.7 mm. We applied a Feldkamp algorithm to reconstruct the micro-CT image. By measuring the presampled modulation transfer function with a 30 microm tungsten thread, we estimated that the micro-CT has a resolution of 5 mm(-1) when the field of view was 6.5 cm. Our results indicate the uniformity of the transaxial micro-CT image and the contrast-to-noise ratio was measured as 1.95 for a radiation dose of 1 cGy. A non-image-based method was employed for merging images from the two imaging modalities. A nude mouse with DiR-BOA, imaged ex vivo, was used to validate the feasibility of the dual-modality system.

Simultaneous fluorescence yield and lifetime tomography from time-resolved transmittances of small-animal-sized phantom.

There has been recently a considerable interest in simultaneously reconstructing yield and lifetime distributions of fluorescent imaging agents inside a bulky tissue, since combined monitoring of these two parameters provides a potential means of in vivo interrogating quantitative and environmental information of specific molecules, as well as accessing interactions among them. It is widely accepted that an advantageous way of accomplishing the task in the context of small-animal imaging is to use a time-domain (TD) modality. In this paper, we present a full three-dimensional, featured-data algorithm for TD diffuse fluorescence tomography, which inverts the Laplace-transformed TD coupled photon diffusion equations and employs a pair of real-domain transform-factors to effectively separate the fluorescent yield and lifetime parameters. By use of a specifically designed 16x16 channel time-correlated single photon counting system and a normalized Born formulation for the inversion, the proposed scheme in a transmission mode is experimentally validated to achieve simultaneous reconstruction of the fluorescent yield and lifetime distributions with reasonable accuracy.

Time-gated perturbation Monte Carlo for whole body functional imaging in small animals.

This paper explores a time-resolved functional imaging method based on Monte Carlo model for whole-body functional imaging of small animals. To improve the spatial resolution and quantitative accuracy of the functional map, a Bayesian hierarchical method with a high resolution spatial prior is applied to guide the optical reconstructions. Simulated data using the proposed approach are employed on an anatomically accurate mouse model where the optical properties range and volume limitations of the diffusion equation model exist. We investigate the performances of using time-gated data type and spatial priors to quantitatively image the functional parameters of multiple organs. Accurate reconstructions of the two main functional parameters of the blood volume and the relative oxygenation are demonstrated by using our method. Moreover, nonlinear optode settings guided by anatomical prior is proved to be critical to imaging small organs such as the heart.

Photoacoustic tomography of small animal brain with a curved array transducer.

We present the application of a curved array photoacoustic tomographic imaging system that can provide rapid, high-resolution photoacoustic imaging of small animal brains. The system is optimized to produce a B-mode, 90-deg field-of-view image at sub-200-microm resolution at a frame rate of approximately 1 frame/second when a 10-Hz pulse repetition rate laser is employed. By rotating samples, a complete 360-deg scan can be achieved within 15 s. In previous work, two-dimensional (2-D) ex vivo mouse brain cortex imaging has been reported. We report three-dimensional (3-D) small animal brain imaging obtained with the curved array system. The results are presented as a series of 2-D cross-sectional images. Besides structural imaging, the blood oxygen saturation of the animal brain cortex is also measured in vivo. In addition, the system can measure the time-resolved relative changes in blood oxygen saturation level in the small animal brain cortex. Last, ultrasonic gel coupling, instead of the previously adopted water coupling, is conveniently used in near-real-time 2-D imaging.

Registration of planar bioluminescence to magnetic resonance and x-ray computed tomography images as a platform for the development of bioluminescence tomography reconstruction algorithms.

The procedures we propose make possible the mapping of two-dimensional (2-D) bioluminescence image (BLI) data onto a skin surface derived from a three-dimensional (3-D) anatomical modality [magnetic resonance (MR) or computed tomography (CT)] dataset. This mapping allows anatomical information to be incorporated into bioluminescence tomography (BLT) reconstruction procedures and, when applied using sources visible to both optical and anatomical modalities, can be used to evaluate the accuracy of those reconstructions. Our procedures, based on immobilization of the animal and a priori determined fixed projective transforms, should be more robust and accurate than previously described efforts, which rely on a poorly constrained retrospectively determined warping of the 3-D anatomical information. Experiments conducted to measure the accuracy of the proposed registration procedure found it to have a mean error of 0.36+/-0.23 mm. Additional experiments highlight some of the confounds that are often overlooked in the BLT reconstruction process, and for two of these confounds, simple corrections are proposed.

Effects of sampling strategy on image quality in noncontact panoramic fluorescence diffuse optical tomography for small animal imaging.

Fluorescence diffuse optical tomography is an emerging technology for molecular imaging with recent technological advances in biomarkers and photonics. The introduction of noncontact imaging methods enables very large-scale data acquisition that is orders of magnitude larger than that from earlier systems. In this study, the effects of sampling strategy on image quality were investigated using an imaging phantom mimicking small animals and further analyzed using singular value analysis (SVA). The sampling strategy was represented in terms of a number of key acquisition parameters, namely the numbers of sources, detectors, and imaging angles. A number of metrics were defined to quantitatively evaluate image quality. The effects of acquisition parameters on image quality were subsequently studied by varying each of the parameters within a reasonable range while maintaining the other parameters constant, a method analogue to partial derivative in mathematical analysis. It was found that image quality improves at a much slower rate if the acquisition parameters are above certain critical values (approximately 5 sources, approximately 15 detectors, and approximately 20 angles for our system). These critical values remain virtually the same even if other acquisition parameters are doubled. It was also found that increasing different acquisition parameters improves image quality with different efficiencies in terms of the number of measurements: for a system characterized by a smaller threshold in SVA (less than 10(-5) in our study), the number of sources is the most efficient, followed by the number of detectors and subsequently the number of imaging angles. However, for systems characterized by a larger threshold, the numbers of sources and angles are equally more efficient than the number of detectors.

A three-dimensional multispectral fluorescence optical tomography imaging system for small animals based on a conical mirror design.

We have developed a three dimensional (3D) multispectral fluorescence optical tomography small animal imaging system with an innovative geometry using a truncated conical mirror, allowing simultaneous viewing of the entire surface of the animal by an EMCCD camera. A conical mirror collects photons approximately three times more efficiently than a flat mirror. An x-y mirror scanning system makes it possible to scan a collimated excitation laser beam to any location on the mouse surface. A pattern of structured light incident on the small animal surface is used to extract the surface geometry for reconstruction. A finite element based algorithm is applied to model photon propagation in the turbid media and a preconditioned conjugate gradient (PCG) method is used to solve the large linear system matrix. The reconstruction algorithm and the system feasibility are evaluated by phantom experiments. These experiments show that multispectral measurements improve the spatial resolution of reconstructed images. Finally, an in vivo imaging study of a xenograft tumor in a mouse shows good correlation of the reconstructed image with the location of the fluorescence probe as determined by subsequent optical imaging of cryosections of the mouse.

A time domain fluorescence tomography system for small animal imaging.

We describe the application of a time domain diffuse fluorescence tomography system for whole body small animal imaging. The key features of the system are the use of point excitation in free space using ultrashort laser pulses and noncontact detection using a gated, intensified charge-coupled device (CCD) camera. Mouse shaped epoxy phantoms, with embedded fluorescent inclusions, were used to verify the performance of a recently developed asymptotic lifetime-based tomography algorithm. The asymptotic algorithm is based on a multiexponential analysis of the decay portion of the data. The multiexponential model is shown to enable the use of a global analysis approach for a robust recovery of the lifetime components present within the imaging medium. The surface boundaries of the imaging volume were acquired using a photogrammetric camera integrated with the imaging system, and implemented in a Monte-Carlo model of photon propagation in tissue. The tomography results show that the asymptotic approach is able to separate axially located fluorescent inclusions centered at depths of 4 and 10 mm from the surface of the mouse phantom. The fluorescent inclusions had distinct lifetimes of 0.5 and 0.95 ns. The inclusions were nearly overlapping along the measurement axis and shown to be not resolvable using continuous wave (CW) methods. These results suggest the practical feasibility and advantages of a time domain approach for whole body small animal fluorescence molecular imaging, particularly with the use of lifetime as a contrast mechanism.

Curved array photoacoustic tomographic system for small animal imaging.

We present systematic characterization of a photoacoustic imaging system optimized for rapid, high-resolution tomographic imaging of small animals. The system is based on a 128-element ultrasonic transducer array with a 5-MHz center frequency and 80% bandwidth shaped to a quarter circle of 25 mm radius. A 16-channel data-acquisition module and dedicated channel detection electronics enable capture of a 90-deg field-of-view image in less than 1 s and a complete 360-deg scan using sample rotation within 15 s. Measurements on cylindrical phantom targets demonstrate a resolution of better than 200 microm and high-sensitivity detection of 580-microm blood tubing to depths greater than 3 cm in a turbid medium with reduced scattering coefficient mu(s) (')=7.8 cm(-1). The system is used to systematically investigate the effects of target size, orientation, and geometry on tomographic imaging. As a demonstration of these effects and the system imaging capabilities, we present tomographic photoacoustic images of the brain vasculature of an ex vivo mouse with varying measurement aperture. For the first time, according to our knowledge, resolution of sub-200-microm vessels with an overlying turbid medium of greater than 2 cm depth is demonstrated using only intrinsic biological contrast.

Quantitative diffuse optical tomography for small animals using an ultrafast gated image intensifier.

The quantitative accuracy of fluorescence and bioluminescence imaging of small animals can be improved by knowledge of the in situ optical properties of each animal. Obtaining in situ optical property maps is challenging, however, due to short propagation distances, requirements for high dynamic range, and the need for dense spatial, temporal, and spectral sampling. Using an ultrafast gated image intensifier and a pulsed laser source, we have developed a small animal diffuse optical tomography system with multiple synthetic modulation frequencies up to >1 GHz. We show that amplitude and phase measurements with useful contrast-to-noise ratios can be obtained for modulation frequencies over the range of approximately 250 to 1250 MHz. Experiments with tissue simulating phantoms demonstrate the feasibility of reconstructing the absorption and scattering optical properties in a small animal imaging system.

A comparison between a time domain and continuous wave small animal optical imaging system.

We present a phantom study to evaluate the performance of the eXplore Optix (Advanced Research Technologies-GE Healthcare), the first commercially available time-domain tomography system for small animal fluorescence imaging, and compare its capabilities with the widely used IVIS 200 (Xenogen Corporation-Caliper) continuous wave planar imaging system. The eXplore Optix, based on point-wise illumination and collection scheme, is found to be a log order more sensitive with significantly higher detection depth and spatial resolution as compared with the wide-area illumination IVIS 200 under the conditions tested. A time-resolved detection system allows the eXplore Optix to measure the arrival time distribution of fluorescence photons. This enables fluorescence lifetime measurement, absorption mapping, and estimation of fluorescent inclusion depth, which in turn is used by a reconstruction algorithm to calculate the volumetric distribution of the fluorophore concentration. An increased acquisition time and lack of ability to image multiple animals simultaneously are the main drawbacks of the eXplore Optix as compared with the IVIS 200.

Simultaneous in vivo dynamic magnetic resonance-diffuse optical tomography for small animal imaging.

We present simultaneous measurement of enhancement kinetics of an optical and a magnetic resonance (MR) contrast agent in a small animal breast tumor model (R3230 ac) using a combined MR-diffuse optical tomographic (MR-DOT) imaging system. A mixture of a small molecular-weight MR contrast agent gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA) and a large molecular-weight optical contrast agent indocyanine green (ICG) was administered intravenously for multimodal dynamic imaging. Coregistration of optical and MR images was accomplished using agar-water-based markers. Using T(2) and dynamic T(1) weighted MR images, we divided the entire tumor into two regions of interest (ROI): a viable and a nonviable region. The absorption enhancements in the ROIs were calculated. An enhancement of the ICG was observed in the viable region. On the contrary, there was a lower enhancement in the nonviable region.