Non-invasive monitoring of commonly used intraocular drugs against endophthalmitis by Raman spectroscopy.

PURPOSE: To develop a non-contact and non-invasive method for quantification of the local concentration of certain antibiotic and antifungal drugs in the eye. STUDY DESIGN/MATERIALS AND METHODS: An integrated CCD-based Raman spectroscopic system designed specifically for ophthalmic applications was used to non-invasively detect the presence of ceftazidime and amphotericin B in ocular media. Specific Raman signatures of the above named drugs were determined for various concentrations that were injected through a needle in the aqueous humor of rabbit eyes in vivo. Raman spectra were subsequently acquired by focusing an argon laser beam within the anterior chamber of the eye. RESULTS: Compared to ocular tissue, unique spectral features of ceftazidime appeared near 1,028, 1,506, 1,586, and 1,641 cm(-1). Amphotericin B exhibited its characteristic peaks at 1,156.5 and 1,556 cm(-1). The amplitude of the spectral peak corresponding to these drugs (acquired by 1 second exposure time and 25 mW of laser power) were determined to be linearly dependent on their local concentration in the anterior chamber of the eye. CONCLUSIONS: Raman spectroscopy may offer an effective tool to non-invasively assess the local concentration of the delivered drugs within the ocular media. This technique potentially could be used to investigate the pharmacokinetics of intraocular drugs in vivo either from a releasing implant or a direct injection.

Noninvasive detection of ganciclovir in ocular tissue by Raman spectroscopy: implication for monitoring of drug release.

UNLABELLED: Treatment of various pathological conditions in ophthalmology, such as cytomegalovirus (CMV) retinopathy and endophthalmitis, requires a local drug intervention rather than a systemic approach. Accurate knowledge of intraocular drug concentration can permit the ophthalmologist to maintain drug levels within the therapeutic levels necessary for an optimal prognosis, while preventing or minimizing toxicity associated with drug overdose. PURPOSE: To develop a noninvasive/noncontact method for quantification of the local concentration of ganciclovir in the ocular media. METHODS: An integrated CCD-based Raman spectroscopic system designed specifically for ophthalmic applications was used to noninvasively detect the presence of ganciclovir in the ocular media. Various known concentrations of ganciclovir were injected into the aqueous humor of rabbit eyes in a pilot study, in vivo. Raman spectra were then acquired by focusing an argon laser beam within the anterior chamber of the eye. The specific Raman signature of ganciclovir was assessed at several concentrations. RESULTS: Spectral features unique to ganciclovir were identified and distinguished from those of ocular tissue. The amplitudes of the spectral peaks corresponding to ganciclovir exhibited a linear dependence on the local concentration of the drug in the anterior chamber of the eye. CONCLUSION: Raman spectroscopy may offer an effective tool for the noninvasive assessment of the local concentration of ganciclovir in the ocular media. This technique offers the potential to determine both the amount and the rate of the drug release from implants designed to deliver antiviral drugs locally within the eye. The availability of such data could enable the ophthalmologist to improve treatment efficacy by avoiding premature or late surgical replacement of the implants.

Raman spectroscopy in ophthalmology: from experimental tool to applications in vivo.

Raman spectroscopy is a qualitative and quantitative optical technique for determining the molecular composition of matter. Improvements in spectroscopic instruments, especially the modality to detect low light level signals extended the Raman technique to biomedical applications, even in delicate structures like the eye. The purpose of this paper was to make an inventory of performed applications of Raman spectroscopy in biomedical science and especially in ophthalmology. A literature search was done using Medline, Current Contents, a patent server on the Internet, and references found in articles and patents. This search revealed a variety of Raman techniques and applications in biomedical research, and an increasing flow of articles starting in the late 1970s on Raman spectroscopy in ophthalmology. This increase in literature about Raman spectroscopy in ophthalmology feeds the expectation that this valuable technique will be introduced in the future into clinical practice.

Noninvasive Raman spectroscopic identification of intraocular lens material in the living human eye.

PURPOSE: To develop a safe noninvasive technique for identifying the material of intraocular lenses (IOLs) implanted in patients. SETTING: Center for Biomedical Engineering and the Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas, USA. METHODS: Raman spectroscopy was used to noninvasively identify the type of IOL implanted after previous cataract surgery in 9 eyes of 6 patients who were legally blind as a result of eye disease. Three IOLs were characterized: poly(methyl methacrylate) (PMMA) (n = 5), acrylic (n = 3), and silicone (n = 1). Confocal Raman spectroscopy was used with a laser power of 95 microW and exposure time of 1 second. RESULTS: Distinct spectral peaks associated with each type of IOL were obtained. These included spectra peaks at 2840 cm(-1), 2946 cm(-1), and 3000 cm(-1) for PMMA; 2917 cm(-1), 2939 cm(-1), and 3055 cm(-1) for acrylic; and 2900 cm(-1), 2961 cm(-1), and 3048 cm(-1) for silicone. The procedure was well accepted by patients, and there were no complications. CONCLUSIONS: The specific Raman spectra of the IOLs allowed for noninvasive determination of IOL material with the use of a safe light dose and an exposure time of 1 second.

Tailoring of new polymeric biomaterials for the repair of medium-sized corneal perforations.

The aim of this study was to investigate whether polymeric biomaterials can be designed such that they become suitable for surgical closure of medium-sized perforations in the cornea, the transparent tissue in the front of the eye. Such a biomaterial must meet stringent requirements in terms of hydrophilicity, strength, transparency, flexibility, and biocompatibility. Four different copolymers of n-butyl methacrylate (BMA) and hexa(ethylene glycol) methacrylate (HEGMA) were prepared and characterized. Poly(BMA) was made as a reference material. Physicochemical properties were measured (contact angles, glass-transition temperatures, thermal degradation, water uptake and swelling), and cytotoxicity in vitro was assessed with a MTT test. Moreover, the interaction between the materials and cultured human corneal epithelial cells was studied. The copolymers may be useful for temporary closure of corneal perforations.

Review and classification of corneal topographers.

Corneal topography has, due to developments in refractive surgery and contact lens fitting, become a widely used diagnostic tool. Many types of topographers have been introduced, but there is some confusion on classification and subsequent principal possibilities of the various devices offered to the practitioner. The purpose of the study reported here was to make an inventory of developed devices, analyse the basic principles and create a classification based on optical principles. A literature search was done using Medline, the IBM Patent Server, and references found in articles and patents. This search resulted in a variety of descriptions that could be classified into 12 groups according to their use of light source and light-matter interaction of which four groups have representatives on the commercial market. This classification can be used by researchers and practitioners to gain insight into the possibilities of a given device in relation to the desired application.

Noninvasive assessment of the hydration gradient across the cornea using confocal Raman spectroscopy.

PURPOSE: The feasibility of Raman spectroscopy for the noninvasive assessment of axial corneal hydration was investigated. METHODS: A scanning confocal Raman spectroscopy system, with an axial resolution of 50 microns, was used to assess noninvasively the water (OH-bond) to protein (CH-bond) ratio as a measure of the hydration in collagen-based phantom media and rabbit corneas. RESULTS: Raman spectra with high signal-to-noise ratios were obtained under in vitro and in vivo conditions within a range of corneal hydration (H = 0.0-8.3 mg water/mg dry wt). The Raman intensity ratio OH/CH showed a strong correlation with the hydration of the phantom medium (R2 > 0.99) and the rabbit corneas (R2 > 0.95). A degree of reproducibility was seen in measurements performed at a specific depth within the cornea (SD = 1.2%-2.7%). Quantitatively, the spatially resolved corneal water content, as assessed with our method, showed an increasing gradient from the anterior to the posterior region, with a difference of approximately 0.9. Significant qualitative differences in the axial hydration gradient were observed between the in vitro and in vivo situation, caused by the presence of an intact tear-film in vivo. Characterization of the axial corneal hydration using Raman spectroscopy provided a reliable estimation of total corneal hydration compared with conventional measurements using pachymetry and lyophilization. CONCLUSIONS: The proposed noninvasive confocal Raman spectroscopic technique has the potential to assess the axial corneal water gradient with a degree of sensitivity and reproducibility.

Development of a wide field height eye topographer: validation on models of the anterior eye surface.

PURPOSE: The aim of this research was to develop a corneal topographer that determines the shape of the entire anterior surface of an eye without assumptions, and with uniformly high accuracy in the center and periphery. METHODS: Based upon a double projection of two sine wave gratings and analysis of the distortion of the sine wave gratings due to the corneal-scleral shape, point-by-point measurements of surface elevation were obtained with a sample density equal to the pixel density of the CCD-detector. Using this principle, a prototype topographer, called the Maastricht Shape Topographer (MST), was developed. The accuracy and reproducibility of the instrument were evaluated using bispheric models of the anterior surface of the eye. RESULTS: The average accuracy of height measurements was +/- 0.55 micron in the 10-mm central area and +/- 22.50 microns in the periphery (14 to 19 mm). Reconstruction accuracy of the radius of curvature was +/- 0.0155 mm (+/- 0.88 D) in the center and +/- 0.0313 mm in the periphery (sclera). Average height reproducibility standard error was 0.0282 micron in the center and 2.6156 microns in the periphery. CONCLUSIONS: With the MST, unambiguous shape measurements of the entire anterior surface of the eye are possible, with accuracy up to clinically accepted standards. MST is able to measure height over a wide area of 20 mm, with a 6-mm depth of field. The tested prototype of the device can be further improved by the use of custom-made optics in order to increase signal to noise ratio in the periphery of the image. This height topographer could offer a reliable method in cases where shape is of paramount importance, e.g., in (scleral) contact lens fitting and refractive surgery.

Yield of fluorescence from indocyanine green in plasma and flowing blood.

The purpose of this work was to obtain more quantitative knowledge about the yield of fluorescence from retinal vessels during indocyanine green angiography (ICG). The yield of fluorescence from blood was investigated for various shear rates, concentrations of ICG, and layer thicknesses. Measurements were performed in vitro on samples of human blood in a cone-plate shear chamber using frontal illumination as in scanning laser angiography. In blood and in plasma, the yield of fluorescence of ICG increased with concentration up to 0.05 and 0.1 mg/ml, respectively. At higher concentrations, the yield decreased for all layer thicknesses. For increasing layer thicknesses, both in plasma and in blood, the yield of ICG fluorescence increased nonlinearly for concentrations higher than 0.012 mg/ml. Saturation occurred for layers thicker than 200 microns in combination with ICG concentrations of 0.4 mg/ml and higher. Application of shear rates within the physiological range of the microcirculation (88/sec and 528/sec) increased the yield of fluorescence from the blood sample compared with stasis. The high transparency of blood for the excitation and emission light of ICG that was demonstrated will lead to superposition of fluorescence from superficial and deeper layers. This superposition precludes quantitative indocyanine angiography of ocular vessels.

Annexin V perturbs or stabilises phospholipid membranes in a calcium-dependent manner.

The potency of annexin V to transport Ca2+ ions across phospholipid membranes was investigated, using large unilamellar phospholipid vesicles loaded with the Ca2+ indicator fura-2. It was demonstrated that annexin V leaves the vesicle membranes intact when added in the presence of 1 mM Ca2+. However, if the vesicles were first incubated with annexin V in the absence of Ca2+, subsequent addition of Ca2+ produced a fluorescence signal due to binding of Ca2+ to fura-2. Centrifugation of the vesicle suspension immediately thereafter showed that this signal originated from the supernatant and not from the sedimented vesicles. Our results show that annexin V causes loss of vesicle integrity in the absence of Ca2+, and leakage of trapped fura-2, rather than inward Ca2+ transport. Bovine serum albumin or Ca2+ concentrations higher than 2.5 mM also caused such fura-2 leakage. Apparently, calcium-dependent binding of annexin V to the membrane prevents aspecific membrane damage caused by this protein.

Shear rate and hematocrit dependence of fluorescence from retinal vessels in fluorescein angiography.

The purpose of this work was to obtain more quantitative knowledge about the yield of fluorescence from retinal vessels during fluorescein angiography. The influence of shear rate, concentration of sodium fluorescein, hematocrit, and layer thickness on the yield of fluorescence from blood were investigated. Measurements were performed in vitro on samples of human blood in a cone-plate shear chamber using frontal illumination. Application of physiologically relevant levels of shear (> 88/sec) decreased the yield of fluorescence from the blood sample considerably as compared with stasis. The yield of fluorescence was proportionally related to the logarithm of the sodium fluorescein concentration in blood up to a sodium fluorescein concentration of 1.2 mg/ml. Above that concentration quenching occurred. An increase in layer thickness at a hematocrit of 45% resulted only in an increase of the yield of fluorescence up to a layer thickness of 25 microns. In conclusion, the sodium fluorescein concentration in blood is the only important factor that determines the yield of fluorescence from the larger retinal vessels in the successive phases of the fluorescein angiogram in a subject with a given hematocrit and hemoglobin concentration. The yield of fluorescence from retinal vessels (> 25 microns) is proportionally related to the logarithm of the sodium fluorescein concentration over a broad range of concentrations.

Is closure of open skin wounds in rats accelerated by argon laser exposure?

The influence of linearly polarized argon laser irradiation (lambda = 488 nm and 514.5 nm) on the closure time of standardized open skin wounds was measured in rats. In two separate controlled experiments no acceleration of wound closure by laser irradiation was observed. In the first experiment the wounds were cleaned during the laser treatment period at 1 J/cm2. The second experiment at 4 J/cm2 was without mechanical wound cleaning. The contradictory results reported in the literature and possible influences of wavelength, energy density, and power density are discussed.