Kwang I. Suh

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 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.

Dynamic light scattering particle-size measurements in turbid media

Dynamic light scattering (DLS) particle size measurments are reported for turbid systems. These include particulate dispersions and suspensions of macromolecules in a wide (7 nm - 800 nm) particle size (diameter) range which look as transparent as water and as turbid as milk (turbidity approximately 6 - 7 orders of magnitude higher than water) and samples of lenticular and skin tissues. Our results indicate a significant improvement in achieving non-invasive, compact, robust, and remotely operated DLS probes which are able to push the envelope of DLS utilization to extremely turbid samples of a variety of particle type and size of macromolecules which were hitherto not possible.

Design and characterization of coherent integrated fiber-optic imaging probes

An integrated fiber-optic probe comprising a short length of multimode fiber that is fusion spliced to a monomode optical fiber has been fabricated for imaging and nonimaging applications. The fiber probe, typically 250 µm in diameter, can deliver a focused Gaussian spot approximately 25 µm in diameter at a distance of approximately 500 µm from the tip. Two off-the-shelf graded-index multimode fibers have been used in the fabrication of imaging and nonimaging probes. These integrated probes have considerably improved the spatial resolution of backscatter lensless fiber probes being utilized in the dynamic light-scattering characterization of colloidal suspension.

Integrated fiber optic probe for dynamic light scattering

An integrated fiber optic probe, comprising a monomode optical fiber fusion spliced to a short length of a graded-index multimode fiber, is fabricated for use as a coherent receiver in dynamic light scattering. The multimode fiber is cleaved to provide a gradient-index fiber lens with a focal length of 125 µm and an ƒ-number close to unity. An integrated fiber receiver is used to measure the intensity-intensity autocorrelation data from a 0.05% by weight concentration of an aqueous suspension of polystyrene latex spheres. Analysis of 100 independent data sets indicates that the particle size can be recovered with an accuracy of ±1%.

Instrumentation and calibration protocol for a continuous wave near infrared hemoximeter

Quantification of hemoglobin content in vivo using continuous wave (CW) near infrared spectroscopy requires an accurate calibration of the measuring system. The authors introduce a recently developed instrument and focus their attention on the calibration issue, proposing a calibration procedure specifically designed for their system, but which can be easily generalized for other CW near-infrared systems. In the paper, the most important calibration procedures and the results obtained are discussed in detail

Measurement of Choroidal blood flow in zero gravity

In this paper we present preliminary measurements on the effects of zero gravity environment on the choroidal blood flow on human volunteer subjects. These experiments were conducted, for the first time, on-board a wide body aircraft (KC-135) during parabolic flight trajectories (0g to 2g environment) using a head-mounted miniature laser Doppler flowmeter.

A direct method of particle sizing based on the statistical processing of scattered photons from particles executing Brownian motion

A direct method of determining the mean diameter of particles executing Brownian motion is presented. The temporal coherence of the scattered field from submicroscopic particles illuminated by laser light is a function of both the integration time and the particle diameter. The temporal degree of coherence of the time-averaged scattered intensity decreases as the integration time increases. Statistical processing of the scattered photons leads to a method of particle sizing (average diameter), which circumvents the need for digital autocor-relation or power spectral estimation.

Sizing of colloidal particles and protein molecules in a hanging fluid drop

We report non-invasive particle size measurements of polystyrene latex colloidal particles and bovine serum albumin (BSA) protein molecules suspended in tiny hanging fluid drops of 30 (mu) L volume using a newly designed fiber optic probe. The probe is based upon the principles of the technique of dynamic light scattering (DLS). The motivation for this work comes from growing protein crystals in outer space. Protein crystals have been grown previously in hanging drops in microgravity experiments on-board the space shuttle orbiter. However, obtaining quantitative information on nucleation and growth of the protein crystals in real time has always been a desired goal, but hitherto not achieved. Several protein researchers have shown interest in using DLS to monitor crystal growth process in a droplet, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. We demonstrate that such an experiment is now possible. Our system offers fast (5 seconds) determination of particle size, utilizes safe levels of very low laser power (less than or equal to 0.2 mW), a small scattering volume (approximately 2 multiplied by 10-5 mm3) and high spatial coherence (beta) values. This is a major step forward when compared to currently available DLS systems.