Pinhole is a critical device in single photon confocal microscopy (SPCM) owning to its ability to block the background
noise scattered from back and forth of the focal plane. Without pinhole, the sectioning ability of SPCM will be degraded
and many background noise signals will occurred together with useful signals, and sometimes these bad noises can
submerge the details that we are interested in. However a pinhole with too small diameter will block both background
noises and part of signals and decrease the intensity of the image. Therefore in many cases pinhole size should be
selected carefully. Unfortunately because of constrains in mechanics, a pinhole that can change its size continuously, for
example from 10 μm to 100 μm, is unavailable. For most commercial confocal microscopies, only several discrete
pinhole sizes are provided, such as 10 μm, 30 μm, 60 μm etc. Things will be even harder for some imaging systems
which use the input interface of a single mode fiber as the pinhole of SPCM, and then the pinhole size of these systems
will be fixed, which far limit the optimization of systems’ performance.
In this paper, we design a size-variable pinhole setup that can offer a virtual pinhole with its diameter adjustable, which
includes a physical pinhole (or single mode fiber) and a fine designed zoom relay (ZR) optical system. The
magnification ratio of this ZR can vary smoothly while keeping the conjugation distance unchanged. The aberrations of
the ZR are well balanced and diffraction-limited image performance are obtained so that the virtual pinhole can block
background scattering noise and pass the in-focus signal effectively and accurately. Simulation results are also provided
and discussed.
We have developed a snapshot optical imaging system capable of taking multiple images simultaneously and
sending them to a CCD detector. Using an innovative lens array design, the beam obtained from the fundus camera is
segmented into several different images passed through several different bandpass filters. Each bandpass filter defines a
unique spectral region of imaging. The images are taken simultaneously into a large silicone chip with a dynamic range
of 16 bits (highly sensitive) and are integrated with a single optical connection to a digital fundus camera.
Our algorithm maps blood oxygen saturation of the retina using several wavelengths. These wavelengths are
capable of approximating the whole hemoglobin spectrum and have been found from a previously developed
hyperspectral algorithm. They include four isosbestic points (522, 548, 569, and 586 nm) and three oxygen-sensitive
points (542, 560, and 586 nm) where the difference between fully oxygenated and deoxygenated blood is at a maximum.
Using MatLab code, color maps of oxygen saturation are produced. The average value taken from all vein areas was 60.53%, assuming that the artery oxygen saturation value is
98%. Oxygen saturation of the tissue was 75.78%. Oxygen saturations of the temporal/inferior/nasal veins ranged from
61.86% to 63.37%; the superior vein was significantly lower (54.19%). Tissue oxygen saturations in different quadrants
of the eye ranged from 74.17% to 76.74%.
Our algorithm has been developed for measuring oxygen saturation of the retina clinically. This was done for
one subject only; further work can extend the measurements to different pigments.
The challenge of correctly identifying malaria infection continues to impede our efforts to control
this disease. Recent studies report highly specific retinal changes in severe malaria patients; these retinal
changes may represent a very useful diagnostic indicator for this disease. To further explore the ocular
manifestations of malaria, we used hyperspectral imaging to study retinal changes caused by Plasmodium
berghei ANKA parasitization in a mouse model.
We collected the spectral reflectance of the ocular fundus from hyperspectral images of the
mouse eye. The blood oxygen sensitive spectral region was normalized for variances in illumination, and
used to calculate relative values that correspond to oxygenated hemoglobin levels. Oxygen hemoglobin
levels are markedly lower in parasitized mice, indicating that hemoglobin digestion by P. berghei may be
detected using spectral reflectance. Furthermore, the ocular reflectance of parasitized mice was
abnormally elevated between 660nm and 750nm, suggesting fluorescence in this region. While the
source of this fluorescence is not yet clear, its presence correlates strongly with P. Berghei parasitization,
and may indicate the presence of hemozoin deposits in the retinal vasculature.
The pathology of severe malaria still presents many questions for clinicians and scientists, and
our understanding of cerebral malaria has been generally confined to clinical observation and postmortem
examination. As the retina represents a portion of the central nervous system that can be easily
examined noninvasively, our technique may provide the basis for an automated tool to detect and examine
severe malaria via retinal changes.
We have developed a snapshot fiber bundle technique that circumvents the issue of saccades of the non-immobilized eye.
In this technology, 458 individual fibers are assembled in a two-dimensional array where each fiber represents a portion
of the image. These fibers are redistributed into two separate one-dimensional fiber rows interfaced into a two-slit
spectrometer. The light from each fiber is decomposed into its spectral components by the spectrometer. Using this
innovative technology, we have been able to detect the whole spectrum of hemoglobin using the single light exposure
capabilities of a fundus camera. The hemoglobin signature of the retinal arteries, veins, and retina tissue can be recorded.
The final result is a complete, 3-dimensional representation of the spectral and spatial information from a single exposure
of the patient. By adjusting the field of view on the imaging portion of the fundus camera, the fiber optic cable may
encompass a larger area. However, this causes a decrease in spatial resolution, so we increased the area of the fiber array
by increasing the number of the fibers from 458 to 648, increased the size of each individual fiber from 10 μm to 20 &μm,
and increased the number of slits to four.
The purpose of this study is to explore the possibility that oxygen (O2) diffusion out of the retinal artery (RA) can
explain the observed significant (P<0.001) decrease of oxygen saturation (O2Sat) in RA as intraocular pressure (IOP) is
raised from 10 to 55 mmHg. Hyperspectral image data from normal monkeys' optic nerve head (ONH) and overlying
retinal blood vessels were recorded at IOP settings of 10, 30, 45 and 55 mmHg. Average percent O2Sat values of the
RAs were found from the recorded blood spectra by comparing to reference spectra from saturated and desaturated red
cell suspensions. Percent O2Sat of the RAs was 78.9% at IOP of 10 mmHg. This decreased to 74.1% at 45 mmHg
(P=0.01); and further decreased to 51.5% at IOP = 55 mmHg (P<0.0001). To interpret these results, we developed a
simple diffusion model assuming that the RA is surrounded by tissues in equilibrium with venous oxygen tension (PO2).
O2 flux across the arterial wall was calculated by Fick's law. The percentage of O2 diffusing out of the RA were 0.6% at
IOP of 10 mmHg, and 38% at IOP of 55 mmHg. Confirmation still requires measurement of blood velocity.
Background and Objective: To evaluate the effect of an acute elevated intraocular pressure (IOP) on oxygen saturation
of structures of the optic nerve head.
Study Design/Materials and Methods: In the cynomolgus monkey eye, IOP was set to 10 mm Hg, and then raised to
30, 45, and 55 mm Hg. The ONH and overlying vessels were imaged using a fundus camera attached to a hyperspectral
imaging system (HSI) at 10 and 30 minutes after IOP elevation. Results: Raising IOP from 10 to 30 mm Hg did not
significantly (P < 0.0001) change saturation in vessels or ONH tissue structures but at 55 mm Hg, all structures showed
significant reduction. Conclusions: Quantitative assay of the blood oxygen saturation in structures on the surface and
overlying the optic nerve head is possible using hyperspectral imaging techniques.
A novel multi-modality optic nerve head image fusion approach has been successfully designed. The new approach has
been applied on three ophthalmologic modalities: angiogram, fundus, and oxygen saturation retinal optic nerve head
images. It has achieved an excellent result by giving the visualization of fundus or oxygen saturation images with a
complete angiogram overlay. During this study, two contributions have been made in terms of novelty, efficiency, and
accuracy. The first contribution is the automated control point detection algorithm for multi-sensor images. The new
method employs retina vasculature and bifurcation features by identifying the initial good-guess of control points using
the Adaptive Exploratory Algorithm. The second contribution is the heuristic optimization fusion algorithm. In order to
maximize the objective function (Mutual-Pixel-Count), the iteration algorithm adjusts the initial guess of the control
points at the sub-pixel level. A refinement of the parameter set is obtained at the end of each loop, and finally an optimal
fused image is generated at the end of the iteration. It is the first time that Mutual-Pixel-Count concept has been
introduced into biomedical image fusion area. By locking the images in one place, the fused image allows
ophthalmologists to match the same eye over time and get a sense of disease progress and pinpoint surgical tools. The
new algorithm can be easily expanded to human or animals' 3D eye, brain, or body image registration and fusion.
Purpose: To develop a multi-spectral method to measure oxygen saturation of the retina in the human eye.
Methods: Five Cynomolgus monkeys with normal eyes were anesthetized with intramuscular ketamine/xylazine and intravenous pentobarbital. Multi-spectral fundus imaging was performed in five monkeys with a commercial fundus camera equipped with a liquid crystal tuned filter in the illumination light path and a 16-bit digital camera. Recording parameters were controlled with software written specifically for the application. Seven images at successively longer oxygen-sensing wavelengths were recorded within 4 seconds. Individual images for each wavelength were captured in less than 100 msec of flash illumination. Slightly misaligned images of separate wavelengths due to slight eye motion were registered and corrected by translational and rotational image registration prior to analysis. Numerical values of relative oxygen saturation of retinal arteries and veins and the underlying tissue in between the artery/vein pairs were evaluated by an algorithm previously described, but which is now corrected for blood volume from averaged pixels (n > 1000). Color saturation maps were constructed by applying the algorithm at each image pixel using a Matlab script.
Results: Both the numerical values of relative oxygen saturation and the saturation maps correspond to the physiological condition, that is, in a normal retina, the artery is more saturated than the tissue and the tissue is more saturated than the vein. With the multi-spectral fundus camera and proper registration of the multi-wavelength images, we were able to determine oxygen saturation in the primate retinal structures on a tolerable time scale which is applicable to human subjects.
Conclusions: Seven wavelength multi-spectral imagery can be used to measure oxygen saturation in retinal artery, vein, and tissue (microcirculation). This technique is safe and can be used to monitor oxygen uptake in humans.
This work is original and is not under consideration for publication elsewhere.
We present an automated method to perform accurate, rapid, and objective measurement of the blood oxygen saturation over each segment of the retinal vascular hierarchy from dual-wavelength fundus images. Its speed and automation (2 s per entire image versus 20 s per segment for manual methods) enables detailed level-by-level measurements over wider areas. An automated tracing algorithm is used to estimate vessel centerlines, thickness, directions, and locations of landmarks such as bifurcations and crossover points. The hierarchical structure of the vascular network is recovered from the trace fragments and landmarks by a novel algorithm. Optical densities (OD) are measured from vascular segments using the minimum reflected intensities inside and outside the vessel. The OD ratio (ODR=OD600/OD570) bears an inverse relationship to systemic HbO2 saturation (SO2). The sensitivity for detecting saturation change when breathing air versus pure oxygen was calculated from the measurements made on six subjects and was found to be 0.0226 ODR units, which is in good agreement with previous manual measurements by the dual-wavelength technique, indicating the validity of the automation. A fully automated system for retinal vessel oximetry would prove useful to achieve early assessments of risk for progression of disease conditions associated with oxygen utilization.
Oxygenation of the facial skin was evaluated in rosacea using a hyperspectral camera. A portable imaging system utilizing crossed-polarization optics for illumination and recording is described. Relative oxygen saturation was determined from rosacea features and compared with normal skin. Saturation maps and light absorption spectra showed a significant increase in the oxygen saturation of the blood in rosacea-affected skin.
We describe a non-invasive in vivo hyperspectral imaging technique for visualizing the spatial distribution of retina and optic nerve head (ONH) tissue oxygenation. Real time images of the fundus are acquired with continuous wavelengths (410-918 nm) to generate a data cube consisting of one spectral and two spatial dimensions. Reflected light from the one-dimensional (1-D) area of the sample is first passed through a grating and is then imaged onto a 12-bit silicone charge- coupled device (CCD) detector. A scanner then proceeds to the next 1-D area of the sample. Acquired image frames contain 256 spatial pixels and 256 wavelengths along rows and columns. Image sequences are scanned along the perpendicular spatial dimension using the push-broom method, whereby the spectrograph and camera are translated under constant velocity with respect to the fundus camera image over 6.6 mm of travel. This set of acquired images contains the full reflected light spectrum at each pixel of a two dimensional area of the retina and ONH. The system employs a focal plane scanner (FPS) using a linear actuator to provide motion. An algorithm processes spectral information at each pixel to represent the varying spatial distribution of retina and ONH tissue oxygenation. Imaging data are obtained from ONH tissue at both normal intraocular pressure (IOP) and acutely raised IOP.
The aim of this study was to investigate the use of photodynamic therapy (PDT) using tin ethyl etiopurpurin (SnET2) for occluding the choriocapillaris in the eyes of pigmented rabbits. Following intravenous injection of SnET2 (0.5 and 1.0 mg/kg) or lipid emulsion alone, the fundus of pigmented rabbits was irradiated within 5 - 15 minutes of photosensitizer injection using 664 nm light at a fluence of 300 mW/cm2 and light doses of 5 - 20 J/cm2. Fundoscopy, fluorescein angiography (FA), and histology were performed at 1, 14 and 28 days after PDT. Following PDT, closure of choriocapillaris was achieved with light doses as low as 5 J/cm2 (17 seconds). Control eyes of rabbits demonstrated no effect to light irradiation at the parameters tested. PDT with SnET2 was effective in this animal model using low levels of activating light for the treatment of choriocapillaris.
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