Roel J. Erckens

Raman Spectroscopy Studies of Metabolic Concentrations in Aqueous Solutions and Aqueous Humor Specimens

Individual concentrations of glucose, lactic acid, and urea down to 0.1 wt % have been investigated in aqueous solutions by the Raman scattering technique. These measurements have been conducted with three different laser excitation wavelengths at 514.5, 632.8, and 751.8 nm in conjunction with a single-grating spectrometer and a liquid nitrogen-cooled charge-coupled device (CCD). Rabbit and human aqueous humor specimens have also been studied by this method. In addition to a water background, the large fluorescence structure in the Raman spectra resulting from these aqueous humor specimens has been subtracted out by being fitted to a first-degree polynomial. Raman peaks similar to the stretching vibrations of methylene and methyl groups associated with glucose and lactate, respectively, were observed in the human specimens within the region from 2800 to 3050 cm−1. Similar structure was observed from the rabbit specimens. In addition, a Raman peak, indicative of the carbon-nitrogen stretching mode of urea and various amino acids, was observed at 1006 cm−1 in all aqueous humor specimens. However, some of the Raman peaks in the rabbit specimens may have resulted from the euthanasia process.

Raman Spectroscopy for Non-Invasive Characterization of Ocular Tissue: Potential for Detection of Biological Molecules

The ability of Raman spectroscopy to remotely characterize ocular tissues using a rapid fibre-optic-based system was explored in this study. An argon ion laser operating at 514.5 nm and a charge-coupled-device-based Raman system were used. Raman spectra from aqueous humour specimens of rabbits and humans, as well as spectra from biological molecules obtained through fresh excised rabbit corneas, were obtained. Prominent peaks were observed which qualitatively matched the known locations of biological molecular peaks such as glucose, lactate and urea. In the rabbit aqueous humour spectra, peak positions were also observed indicative of amino acids and proteins. Raman spectra were also obtained from the rabbit lens showing peak positions indicative of amide I and III modes, sulphydryl and CH stretching modes and amino acids. This study demonstrated that it may be possible to characterize ocular tissue structures and to determine in a qualitative manner the presence of various biological molecules in aqueous humour by means of a Raman-based fibre optic instrument.

Monitoring of aqueous humor metabolites using Raman spectroscopy

Laser Raman scattering has been used to monitor glucose and lactate metabolites within aqueous humor specimens obtained from nine human eyes during cataract surgery. Nine postmortem rabbit eyes were also investigated. Raman measurements were obtained using a single grating Raman spectrometer with a liquid nitrogen cooled CCD. A 514.5 nm line from an argon laser was used to illuminate capillaries containing several microliters of aqueous humor. A water background was subtracted from each of the aqueous humor Raman spectra. This experimental system was calibrated so that each metabolite in water could be measured down to 0.1 weight percent. Raman peaks indicative of the stretching vibrations of methylene and methyl groups associated with glucose and lactate, respectively, were observed in the human specimens. A second stretching mode characteristic of lactate between the carbon atom and either the carboxylic acid group or carboxylate ion group was also observed providing a distinguishing feature between the glucose and lactate Raman peaks. Similar structure was observed from the rabbit specimens, but these samples have recently been found to have been contaminated during euthanasia.

Confocal Raman spectroscopy system for noncontact scanning of ocular tissues: an in vitro study

A long-working-distance fiber-optic-based confocal Raman spectroscopy (CRS) system, operating in the backscatter mode, was developed for rapid noninvasive characterization of ocular tissue. In vitro near-real-time axial scanning through ocular tissue was achieved using a CCD camera and a high-numerical-aperture long-working-distance microscope objective in a telecentric configuration. The system provides high spatial resolution (20 to 150 µm) of transparent ocular tissues up to 13 mm deep into the eye in a noncontact fashion while utilizing low argon-laser power and rapid scanning times (25 mJ), yielding a SNR range from 30 to 75. To test the performance of the system for characterizing ocular tissue, Raman spectra from rabbit eyes were obtained in vitro. Axial scans of the cornea, the aqueous humor, and the lens provided discrete and specific Raman spectra from each tissue, in both the lower and the higher wave-number region. Characteristic Raman signals common to all tissues are the OH vibrations (1650 and 3100 to 3700 cm-1) and the vibrations corresponding to amino acids (phenylalanine at 1003 cm-1, tryptophan at 760 and 881 cm-1, and tyrosine at 646 cm-1). The ocular lens can be identified by three distinct peaks (aromatic and aliphatic CH stretching and OH bending modes), of which the aromatic CH stretching mode (?3057 cm-1) is lens-specific. The cornea can be identified by the presence of two distinct peaks (aliphatic CH stretching and OH bending) and the absence of the aromatic CH stretching mode. The aqueous humor can be identified by the presence of the OH bending mode and the lack of the both CH stretching modes. A long-working-distance confocal Raman spectroscopy system may offer a novel technique for the noncontact spatially resolved biochemical characterization of various tissue layers of the anterior segment of the eye.

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 &mgr;W 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.

Identification of intraocular lens materials using confocal Raman spectroscopy

PURPOSE To develop and test a noninvasive method to identify intraocular lens (IOL) materials in vitro. SETTING Center for Biomedical Engineering and the Department of Ophthalmology, University of Texas Medical Branch, Galveston, Texas, USA. METHODS A laser confocal Raman spectroscopy system (Conforam) was used for the noninvasive assessment of Raman spectra in the lower and the higher spectral regions (299.1 to 1833.7 cm-1 and 2633.8 to 3819.6 cm-1, respectively) of 4 IOL materials: silicone, poly(methyl methacrylate) (PMMA), acrylic, and hydrogel. RESULTS Each lens material showed a distinctive spectrum in both the higher and the lower spectral regions. Most materials had unique peaks and a distinct profile using 1 mW of laser power and a 1 second exposure time. All materials still had a unique spectrum in both the higher and the lower region that allowed 1 material to be distinguished from the others. CONCLUSIONS A Conforam differentiated silicone, PMMA, acrylic, and hydrogel lenses in vitro. Raman spectroscopy using the Conforam may provide a fast, safe, and reliable noninvasive method to gain information about the material of an implanted IOL and the stability of lens materials and their coatings.

Noninvasive assessment of ocular tissue using confocal Raman spectroscopy

We have developed a confocal Raman spectroscopy system in order to noninvasively characterize ocular tissue with both an in vitro nd in vivo capability. This systems consists of a long working distance optical probe, which focuses the incident laser light on the tissue and collects the backscattered Raman signal, a single grating spectrometer with CCD camera, and an optical fiber which couples the optical probe to the spectrometer. Essential to the confocal design is that the sample volume is limited, preventing detection of interference signals and fluorescence from adjacent ocular tissues. This sample volume is adjustable by changing the diameter of the collection fiber which acts as the pinhole in the system. Potential applications of this technique such as assessing corneal hydration and quantifying pharmacokinetic drug transport across the cornea will be discussed.