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.

Holographic image storage in eu(3+)-doped alkali aluminosilicate glasses.

We demonstrate that holographic information can be stored in Eu(3+)-doped alkali aluminosilicate glasses. The holograms were developed by a two-beam mixing configuration with a write-beam wavelength (465.8 nm) corresponding to the (7)F(0) ? (5)D(0) transition of the Eu(3+) ions. The images were reconstructed either with the wavelength used to record them or with wavelengths below this transition (543.5 and 632.8 nm). We stored clear holographic images using a total writing power of 5 mW and an exposure time of 20 s. In addition, clear holograms were recorded with an exposure time of 200 ms when 100 mW of the writing power was used. The exposure time and the writing power required to obtain clear holographic images are dependent on the Eu(3+) concentration.

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.

Non-invasive assessment of ocular pharmacokinetics using Confocal Raman Spectroscopy.

A Laser Scanning Confocal Raman Spectroscopy (LSCRS) system was applied for the non-invasive quantification of the transport of a drug through the rabbit cornea in vivo. Employing LSCRS, the changes in the amplitude of a drug-specific Raman signal were assessed over time in the tearfilm and corneal epithelium of the living rabbit eye (n = 6), after topical application of 25 microL Trusopt 2%. This allowed for quantification of pharmacokinetic variables. The effect of the drug on corneal hydration was also monitored. LSCRS demonstrated adequate sensitivity and reproducibility, for continuous real-time monitoring of the Trusopt concentration. Each concentration-time curve had a bi-phasic trend; the rapid initial phase (t<8 min.) corresponds to the nonproductive losses of Trusopt from the tears (k10 = 0.24+/-0.04 min(-1), and the slower later phase (t>20 min.) is the result of transfer of the drug from the corneal epithelium to the stroma (k23 = 0.0047+/-0.0004 min(-1). Drug absorption into the corneal epithelium occurred at a rate of k12 = 0.034+/-0.006 min(-1). Trusopt caused an acute dehydrating effect, with a maximum decrease in corneal hydration of approximately 15% at approximately 60 min. following application of the drug. LSCRS has the specificity, sensitivity, reproducibility and spatial resolution for employment as a potentially valuable tool for the study of ocular pharmacokinetics.

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.

Refractive Index Measurements using a CCD.

We measure the refractive index of materials using a CCD camera with a laser beam profiler in the familiar Brewster's angle experiment. This allows us to isolate quickly and accurately the Brewster's angle close to the resolution of the sample rotation stage. The uncertainty in the index of refraction measurement is similar to that of the standard minimum-deviation technique.

Analysis of metabolites in aqueous solutions by using laser Raman spectroscopy.

Laser Raman scattering is conducted on aqueous solutions that contain organic chemicals that include glucose, lactate, ascorbate, pyruvate, and urea. At the concentrations of interest (below 1.0 wt. %), these various metabolites are found to scatter light independently of each other, and the scattering is linearly proportional to their concentrations. Through proper subtraction of water background scattering, the spectrum that is due to metabolite scattering is obtained and the composition of the solution can be determined by fitting its Raman spectrum with a linear sum of the known pure metabolite spectra. The spectrum of rabbit aqueous humor is presented and the potential application of this analytical method, such as noninvasive glucose monitoring, is discussed.