Rafat R. Ansari

Clinical Detection of Precataractous Lens Protein Changes Using Dynamic Light Scattering

OBJECTIVE To use dynamic light scattering to clinically assess early precataractous lens protein changes. METHODS We performed a cross-sectional study in 380 eyes of 235 patients aged 7 to 86 years with Age-Related Eye Disease Study clinical nuclear lens opacity grades 0 to 3.8. A dynamic light-scattering device was used to assess alpha-crystallin, a molecular chaperone protein shown to bind other damaged lens proteins, preventing their aggregation. The outcome measure was the alpha-crystallin index, a measure of unbound alpha-crystallin in each lens. The association of the alpha-crystallin index with increasing nuclear opacity and aging was determined. RESULTS There was a significant decrease in the alpha-crystallin index associated with increasing nuclear lens opacity grades (P < .001). There were significant losses of alpha-crystallin even in clinically clear lenses associated with aging (P < .001). The standard error of measurement was 3%. CONCLUSIONS Dynamic light scattering clinically detects alpha-crystallin protein loss even in clinically clear lenses. alpha-Crystallin index measurements may be useful in identifying patients at high risk for cataracts and as an outcome variable in clinical lens studies. CLINICAL RELEVANCE The alpha-crystallin index may be a useful measure of the protective alpha-crystallin molecular chaperone reserve present in a lens, analogous to creatinine clearance in estimating renal function reserve.

A fiber‐optic probe for particle sizing in concentrated suspensions

A fiber‐optic probe employing two monomode optical fibers, one for transmitting a Gaussian laser beam to the scattering volume and the second, positioned at some backscatter angle, for receiving the scattered light is described. Performance and suitability of the system for a process control environment is assessed by studying a suspension of polystyrene latex particles over a wide range of sizes and concentrations. The results show that the probe is ideal for a process control environment in industrial and laboratory applications. Particle size is recovered, without any additional corrections for multiple light scattering, in concentrations containing up to 10% solids of 39‐nm polystyrene latex spheres.

New optical scheme for a polarimetric-based glucose sensor

A new optical scheme to detect glucose concentration in the aqueous humor of the eye is presented. The ultimate aim is to apply this technique in designing a new instrument for, routinely and frequently, noninvasively monitoring blood glucose levels in diabetic patients without contact (no index matching) between the eye and the instrument. The optical scheme exploits the Brewster reflection of circularly polarized light off of the lens of the eye. Theoretically, this reflected linearly polarized light on its way to the detector is expected to rotate its state of polarization, owing to the presence of glucose molecules in the aqueous humor of a patients eye. An experimental laboratory setup based on this scheme was designed and tested by measuring a range of known concentrations of glucose solutions dissolved in water. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

A fiber optic probe for monitoring protein aggregation, nucleation and crystallization

Protein crystals are often experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth processes in hanging droplets (∼ 30 μL) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (microgravity) protein crystallization system configurations to test its capabilities. These include conventional batch (cuvette or capillary) systems, a hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature-induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (a few μW) and essentially eliminates the usual problems associated with optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system makes possible the exploration of new ways to grow good quality crystals suitable for X-ray crystallographic analysis and may contribute to a concrete scientific basis for understanding the process of crystallization.

Dynamic light scattering of diabetic vitreopathy.

BACKGROUND Diabetes induces pathology throughout the body via nonenzymatic glycation of proteins. Vitreous, which is replete with type II collagen, undergoes significant changes in diabetes. The resultant diabetic vitreopathy plays an important role in diabetic retinopathy. Detecting these molecular changes could provide insight into diabetic eye disease as well as molecular effects elsewhere in the body. METHODS Human eyes were obtained at autopsy and studied in the fresh, unfixed state. Sclera, choroid, and retina were dissected off the vitreous for dark-field slit microscopy and dynamic light scattering (DLS). For the former, the entire vitreous was exposed. For the latter, only a window at the equator was dissected in some specimens, and the anterior segment was removed leaving the posterior lens capsule intact in others. DLS was performed to determine particle sizes at multiple sites 0.5 mm apart, spanning the globe at the equator (window dissections) and along the antero-posterior axis. RESULTS Dark-field slit microscopy in diabetic subjects detected findings typical of age-related vitreous degeneration, but at much younger ages than nondiabetic controls. Noninvasive DLS measurements found a greater heterogeneity and larger particle sizes in vitreous of subjects with diabetes as compared to age-matched controls. CONCLUSIONS DLS can detect and quantify the early molecular effects that cause vitreous collagen fibrils to cross-link and aggregate. This could provide valuable insight into ocular and systemic effects of hyperglycemia, because the molecular changes in diabetic vitreopathy could serve as an index of such effects throughout the body. In addition to the diagnostic implications, this methodology could provide a rapid, reproducible way to monitor the response to therapy with novel agents intended to prevent the complications of diabetes on a molecular level.

Measurement of Lens Protein Aggregation in Vivo Using Dynamic Light Scattering in a Guinea Pig/UVA Model for Nuclear Cataract

The role of UVA radiation in the formation of human nuclear cataract is not well understood. We have previously shown that exposing guinea pigs for 5 months to a chronic low level of UVA light produces increased lens nuclear light scattering and elevated levels of protein disulfide. Here we have used the technique of dynamic light scattering (DLS) to investigate lens protein aggregation in vivo in the guinea pig/UVA model. DLS size distribution analysis conducted at the same location in the lens nucleus of control and UVA‐irradiated animals showed a 28% reduction in intensity of small diameter proteins in experimental lenses compared with controls (P < 0.05). In addition, large diameter proteins in UVA‐exposed lens nuclei increased five‐fold in intensity compared to controls (P < 0.05). The UVA‐induced increase in apparent size of lens nuclear small diameter proteins was three‐fold (P < 0.01), and the size of large diameter aggregates was more than four‐fold in experimental lenses compared with controls. The diameter of crystallin aggregates in the UVA‐irradiated lens nucleus was estimated to be 350 nm, a size able to scatter light. No significant changes in protein size were detected in the anterior cortex of UVA‐irradiated lenses. It is presumed that the presence of a UVA chromophore in the guinea pig lens (NADPH bound to zeta crystallin), as well as traces of oxygen, contributed to UVA‐induced crystallin aggregation. The results indicate a potentially harmful role for UVA light in the lens nucleus. A similar process of UVA‐irradiated protein aggregation may take place in the older human lens nucleus, accelerating the formation of human nuclear cataract.

Ocular static and dynamic light scattering: a noninvasive diagnostic tool for eye research and clinical practice

The noninvasive techniques of static and dynamic light scattering are emerging as valuable diagnostic tools for the early detection of ocular and systemic diseases. These include corneal abnormalities, pigmentary dispersion syndrome, glaucoma, cataract, diabetic vitreopathy, and possibly macular degeneration. Systemic conditions such as diabetes and possibly Alzheimers disease can potentially be detected early via ocular tissues. The current state of development of these techniques for application to ophthalmic research and ultimately clinical practice is reviewed.

Use of Dynamic Light Scattering and Scheimpflug Imaging for the Early Detection of Cataracts

Cataract is a leading cause of blindness. Diabetes and glycemic conditions enhance the chances of developing cataracts early. At clinical stage cataracts are detected and documented by slit-lamp biomicroscopy and Scheimpflug photography. A compact fiber optic probe, developed for space experiments, was mounted on a Scheimpflug imaging system. The probe detects and documents cataracts at pre-clinical stage noninvasively and quantitatively. The early detection at the molecular level may lead to medical treatment of cataracts, better control of glycemia, and diabetes mellitus.

Particle-size and velocity measurements in flowing conditions using dynamic light scattering

The noninvasive optical technique of dynamic light scattering (DLS) is routinely used to characterize dilute and transparent submicrometer particle dispersions in laboratory environments. A variety of industrial and biological applications would, however, greatly benefit from on-line monitoring of dispersions under flowing conditions. We present a model experiment to study flowing dispersions of polystyrene latex particles of varying sizes under varying flow conditions by using a newly developed fiber-optic DLS probe. A modified correlation function proposed in an earlier study is applied to the analysis of extracting the size and velocity of laminar flowing particulate dispersions. The complementary technique of laser Doppler velocimetry is also used to measure the speed of moving particles to confirm the DLS findings.

Hydrogen therapy may reduce the risks related to radiation-induced oxidative stress in space flight

Cosmic radiation is known to induce DNA and lipid damage associated with increased oxidative stress and remains a major concern in space travel. Hydrogen, recently discovered as a novel therapeutic medical gas in a variety of biomedical fields, has potent antioxidant and anti-inflammatory activities. It is expected that space mission activities will increase in coming years both in numbers and duration. It is therefore important to estimate and prevent the risks encountered by astronauts due to oxidative stress prior to developing clinical symptoms of disease. We hypothesize that hydrogen administration to the astronauts by either inhalation or drinking hydrogen-rich water may potentially yield a novel and feasible preventative/therapeutic strategy to prevent radiation-induced adverse events.