António Miguel Morgado

Diabetes and corneal cell densities in humans by in vivo confocal microscopy.

Purpose: Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. Methods: The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. Results: The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 ± 171, 5950 ± 653, and 2690 ± 302 cells/mm2 in controls and 815 ± 260, 5060 ± 301, and 2660 ± 364 cells/mm2 in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 ± 4090, 19,390 ± 3120, and 25,700 ± 3260 cells/mm3 in controls and 27,560 ± 3880, 21,930 ± 2110, and 25,790 ± 3090 cells/mm3 in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (−15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. Conclusions: The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.

Corneal Cell Density Measurement in vivo by Scanning Slit Confocal Microscopy: Method and Validation

Purpose: Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM). Method: A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods. Results: Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from –24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean ± SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 ± 162, 5,817 ± 632 and 2,743 ± 285 cells·mm–2 (surface), and in the anterior, mid and posterior stroma 28,616 ± 3,081, 19,578 ± 4,421 and 26,073 ± 5,962 cells·mm–3 (volumetric). These results are in accordance with those of other investigators. Conclusions: The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

A method for corneal nerves automatic segmentation and morphometric analysis

The segmentation and morphometric analysis of corneal sub-basal nerves, from corneal confocal microscopy images, has gained recently an increased interest. This interest arises from the possibility of using changes in these nerves as the basis of a simple and non-invasive method for early detection and follow-up of peripheral diabetic neuropathy, a major cause of chronic disability in diabetic patients. Here, we propose one method for automatic segmentation and analysis of corneal nerves from images obtained in vivo through corneal confocal microscopy. The method is capable of segmenting corneal nerves, with sensitivity near 90% and a percentage of false recognitions with an average of 5.3%. The nerves tortuosity was calculated and shows statistically significant differences between healthy controls and diabetic individuals, in accordance to what is reported in the literature.

The optical coupling of analog signals

An opto-coupling circuit based on the Siemens IL300 linear optocoupler and the methods to assess its static and dynamic performances are presented. It is shown that IL300, due to its built-in linearizing feedback photodiode, makes it possible to build coupling schemes that associate good linearity with the inherent properties of optical devices, as true galvanic isolation, high isolation voltage and transmission down to DC. The integral linearity, 0.029%, obtained on the coupling of typical nuclear spectroscopy pulses makes us believe that traditional capacitive coupling used in nuclear spectroscopy circuits can be replaced by optical coupling in the near future.

Optical Coherence Tomography: A Concept Review

Optical coherence tomography (OCT) is an imaging modality broadly used in biological tissue imaging. In this chapter, we review the history of OCT and its development throughout the last years. We will focus on the physical concept of OCT imaging of the eye fundus, considering several settings currently used. We also list some research directions of recent and ongoing work concerned with the future developments of the technique and its application.

Automatic corneal nerves recognition for earlier diagnosis and follow-up of diabetic neuropathy

Peripheral diabetic neuropathy is a major cause of chronic disability in diabetic patients. Morphometric parameters of corneal nerves may be the basis of an ideal method for early diagnosis and assessment of diabetic neuropathy. We developed a fully automatic algorithm for corneal nerve segmentation and morphometric parameters extraction. Luminosity equalization was done using local methods. Images structures were enhanced through phase-shift analysis, followed by Hessian matrix computation for structure classification. Nerves were then reconstructed using morphological methods. The algorithm was evaluated using 10 images of corneal nerves, by comparing with manual tracking. The average percent of nerve correctly segmented was 88.5% ± 7.2%. The percent of false nerve segments was 3.9% ± 2.2%. The average difference between automatic and manual nerve lengths was -28.0 ± 30.3 μm. Running times were around 3 minutes. The algorithm produced good results similar to those reported in the literature.

Two-photon spectral fluorescence lifetime and second-harmonic generation imaging of the porcine cornea with a 12-femtosecond laser microscope

Abstract. Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

Low-level laser therapy on skeletal muscle inflammation: evaluation of irradiation parameters

Abstract. We evaluated the effect of different irradiation parameters in low-level laser therapy (LLLT) for treating inflammation induced in the gastrocnemius muscle of rats through cytokines concentration in systemic blood and analysis of muscle tissue. We used continuous (830 and 980 nm) and pulsed illuminations (830 nm). Animals were divided into five groups per wavelength (10, 20, 30, 40, and 50 mW), and a control group. LLLT was applied during 5 days with a constant irradiation time and area. TNF-α, IL-1β, IL-2, and IL-6 cytokines were quantified by ELISA. Inflammatory cells were counted using microscopy. Identical methodology was used with pulsed illumination. Average power (40 mW) and duty cycle were kept constant (80%) at five frequencies (5, 25, 50, 100, and 200 Hz). For continuous irradiation, treatment effects occurred for all doses, with a reduction of TNF-α, IL-1β, and IL-6 cytokines and inflammatory cells. Continuous irradiation at 830 nm was more effective, a result explained by the action spectrum of cytochrome c oxidase (CCO). Best results were obtained for 40 mW, with data suggesting a biphasic dose response. Pulsed wave irradiation was only effective for higher frequencies, a result that might be related to the rate constants of the CCO internal electron transfer process.

Evaluation of a non-invasive fluorescence technique as a marker for diabetic lenses in vivo.

Abstract · Background: A fluorescence spectrometer has been constructed to study in vitro and in vivo fluorescence of human lenses. This instrument can measure fluorescence emission spectra, which can be useful in the characterisation of the lens endogenous fluorophores and evaluation of the feasibility of fluorescence measurement as a non-invasive marker for diabetes. The spectrometer allows determination of the optimum excitation and emission wavelengths, which can be used in simpler instrumentation for monitoring purposes. · Methods: To est the application in such studies a homogeneous group of type II diabetic subjects and normal controls was studied. For each subject the fluorescence emission spectra was measured using a spectrometer prototype consisting of a modified slit lamp coupled to a optical multichannel analyser (OMA). The incorporation of narrow-band filters allows the selection of three different excitation wavelengths: 404 nm, 436 nm and 485 nm. · Results: With both in vitro and in vivo measurements, no significant differences were found between diabetic and normal lenses concerning the wavelength of maximum emission of fluorescence. However, the spectra (λexc=436 nm) between 480 and 550 nm were better defined with diabetic lenses. Using ratios of fluorescence intensity at two different wavelengths allows for good discrimination between normal controls and diabetic patients. The use of ratios largely removes the effects due to attenuation of excitation light and emitted fluorescence. · Conclusions: The non-invasive evaluation of lens fluorescence is proposed as early indicator of ocular complications associated with diabetes.

Corneal nerves segmentation and morphometric parameters quantification for early detection of diabetic neuropathy

Morphological parameters of the corneal sub-basal nerve plexus may be the basis of a simple and noninvasive method for detection and follow-up of diabetic neuropathy. These nerves can be analyzed from images obtained in vivo by corneal confocal microscopy. In this work we present and evaluate an automatic methodology capable of identifying corneal nerves and determine various morphometric parameters.