Susana F. Silva

Diabetic peripheral neuropathy assessment through corneal nerve morphometry

Diabetic peripheral neuropathy is one of the most common complications of diabetes. It affects 50% of the patients after 25 years of disease. Its early diagnosis and accurate assessment are important to define the higher risk patients. A non-invasive technique for its assessment was developed. The technique is based on morphometric parameters of corneal nerves, obtained by analysis of corneal confocal microscopy images of the sub-basal nerve plexus. We examined 12 type-2 diabetic patients (average age: 58±10 years) and 8 healthy controls (54±7 years). We found differences statistically significant for nerve length, density, width and branching parameters, when we compare individuals with and without neuropathy. The corneal sub-basal nerve plexus morphology has the potential for identifying the presence of diabetic peripheral neuropathy and evaluating its severity.

Evaluation of corneal nerves morphology for diabetic peripheral neuropathy assessment

The evaluation of corneal nerve morphology by optical methods may form the basis of a simple, non-invasive technique for early diagnosis and accurate assessment of peripheral diabetic neuropathy. Currently, corneal nerves can be imaged in vivo using corneal confocal microscopy, an expensive technique that is only available at large medical units. Our goal is to develop an optical technique for peripheral neuropathy assessment, through corneal nerves imaging, based on simple, easy to operate and widespread instrumentation. This technique will be built upon automatic algorithms for corneal nerves segmentation and morphometric analysis and an optical confocal module for recording corneal nerves images using a standard slit-lamp, the most commonly used instrument in ophthalmic practice to observe the anterior eye. Here we present the current status and results of this ongoing project.

Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning

Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.

Data acquisition and laser scanning synchronism in SS-OCT — An experimental apparatus

Optical Coherence Tomography (OCT) is a high-resolution, non-invasive and contactless imaging technique based on optical interferometry. After the Time Domain (TD) and the Spatially Encoded Frequency Domain (SEFD), the Swept-Source (SS) OCT, also known as Time Encoded Frequency Domain (TEFD) OCT, is the latest technology available. It is based on a fast wavelength-sweeping of a narrow laser line over the bandwidth of the source. SWEEP TRIGGER and K-CLOCK signals are provided by the laser source and establish the time base for the overall system. Data acquisition and the two-dimensional scanning of the object of interest must be accurately synchronized after those signals. The proposed solution for the overall scanning/acquisition/sweeping synchronism is an integrated and dedicated FPGA-based control system that is being developed and will generate the automatic, programmable and flexible operation that will replace the present, manually adjustable, system. This way, complete reproducibility of experimental conditions will be obtained, along with the possibility of optimizing the galvanometer /mirrors control. This system is being built around a Xilinx XC6SLX45 FPGA and will be integrated in the already developed control software of the OCT system.

Time-gated FLIM microscope for corneal metabolic imaging

Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

Fluorescence lifetime microscope for corneal metabolic imaging

Assessing corneal metabolism may provide clinicians a tool for diagnosing corneal cells dysfunctions prior to its pathological expression. Flavin adenine dinucleotide (FAD), a metabolic co-factor, exhibits two lifetime components (long and short) upon blue light excitation. Due to that, fluorescence lifetime imaging microscopy (FLIM) may provide a method to evaluate corneal cells metabolism non-invasively. We are developing a single-photon, time-gated fluorescence lifetime microscope for in vivo corneal imaging using structured illumination to improve optical sectioning. Single-photon imaging is provided by a picosecond diode laser with emission at 443nm. Structured illumination is implemented by modulating the laser light through a Digital Micromirror Device (DMD). The fluorescence imaging acquisition is based on an ultrafast time-gated intensified CCD camera operating with gates down to 200ps. We present preliminary data regarding the timing and optical performance of the microscope.

Diabetic peripheral neuropathy assessment through texture based analysis of corneal nerve images

Diabetic peripheral neuropathy (DPN) is one common complication of diabetes. Early diagnosis of DPN often fails due to the non-availability of a simple, reliable, non-invasive method. Several published studies show that corneal confocal microscopy (CCM) can identify small nerve fibre damage and quantify the severity of DPN, using nerve morphometric parameters. Here, we used image texture features, extracted from corneal sub-basal nerve plexus images, obtained in vivo by CCM, to identify DPN patients, using classification techniques. A SVM classifier using image texture features was used to identify (DPN vs. No DPN) DPN patients. The accuracies were 80.6%, when excluding diabetic patients without neuropathy, and 73.5%, when including diabetic patients without diabetic neuropathy jointly with healthy controls. The results suggest that texture analysis might be used as a complementing technique for DPN diagnosis, without requiring nerve segmentation in CCM images. The results also suggest that this technique has enough sensitivity to detect early disorders in the corneal nerves of diabetic patients.

Development of a time-gated fluorescence lifetime microscope for in vivo corneal metabolic imaging

Metabolic imaging can be a valuable tool in the early diagnosis of corneal diseases. Cell metabolic changes can be assessed through non-invasive optical methods due to the autofluorescence of metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Both molecules exhibit double exponential fluorescence decays, with well-separated short and long lifetime components, which are related to their protein-bound and free states. Corneal metabolism can be monitored by measuring the relative contribution of these two components. Here we report on the development of a fluorescence lifetime imaging microscope for in vivo measurement of FAD fluorescence lifetimes in corneal cells. The microscope is based on one-photon fluorescence excitation, through a pulsed blue diode laser. Fluorescence lifetime imaging is achieved using the Time-Gated technique. Structured illumination is used to improve the low axial resolution of wide-field time-gated FLIM. A Digital Micromirror Device (DMD) is used to produce the sinusoidal patterns required by structural illumination. The DMD control is integrated with the acquisition software of the imaging system which is based on an ultra-high speed gated image intensifier coupled to a CCD camera. We present preliminary results concerning optical and timing performance of the fluorescence lifetime microscope. Preliminary tests with ex-vivo bovine corneas are also described.

Corneal Nerve Morphometry for Diabetic Peripheral Neuropathy Assessment

Diabetic peripheral neuropathy (DPN) is one of the more common complications of diabetes, being associated to 50-75% of non-traumatic amputations. Early diagnosis of DPN often fails or occurs only when patients became symptomatic due to the non-availability of a simple, reliable, noninvasive method.

Corneal metabolic imaging by FAD autofluorescence lifetime

The final goal of this work is to develop an corneal microscope based on fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-factor FAD. In several corneal pathologies metabolic alterations occur prior to the pathologic expression therefore the assessment of corneal cells metabolic state could be a great advantage. Here, we aim to demonstrate the feasibility of using FAD autofluorescence lifetime to assess corneal cells metabolic state. We found an increase in protein-bound FAD fluorescence lifetime and a decrease in this component relative contribution 48h and 72h after animal sacrifice. These results are in agreement with the expected decrease in cell metabolism which has also been confirmed by MTT assay. Although the results are promising, the number of animals per conditions must be increased and further in-vivo studies are required to fully characterize FAD fluorescence lifetime in corneal tissues.