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.

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.Diabetic peripheral neuropathy,confocal microscopy images, corneal nerve morphology, morphometric parameters. I. CONTEXTDiabetic neuropathy is among the commonest long-term complications of diabetes, representing the main cause of chronic disability in diabetic patients [I]. This condition encompasses a large spectrum of nerve disorders induced by diabetes over time, leading to the development of nerve damage throughout the human body [2]. Nerve complications can easily occur in different organ systems, including the digestive tract and cardiovascular system [3]. Peripheral nerve involvement is extremely frequent in diabetes and it has been revealed that diabetic peripheral neuropathy (DPN) is estimated to be present in approximately 8% of newly diagnosed diabetic patients [3]. In long term, unrevealed and untreated neuropathy is the main cause of foot infections that not heal, foot ulcers followed in many cases by inevitable amputation. DPN affects up to 50% of the patients after 25 years of disease, being associated to 50-75% of non traumatic amputations [3,4]. Master Thesis, IULIAN OTEL Portuguese chapter of IEEE EMBS 3r d Portuguese Meeting in Bioengineering, The prevalence of diabetic neuropathies is dramatically increasing with the enormous burden of type-2 diabetes, but the true occurrence remains unrevealed, with variable reports in diabetic patients depending mostly on the criteria and methods used to identify neuropathy [5, 6]. Frequently, patients with nerve damage have no symptoms, whilst most part have extremely painful symptoms, such as burning pain, squeezing, constricting, freezing, allodynia or non-painful neuropathic deficits, such as asleep, tingling, numbness-loss of feeling in the hands, arms, feet, and legs [3, 5]. Therefore, an early diagnosis and accurate assessment of DPN are extremely important to defme the higher risk patients. However, early diagnosis often fails or occurs only when patients became symptomatic due to the non-availability of a simple reliable non-invasive method. In recent studies numerous researchers proposed the assessment of DPN through corneal morphologic parameters. These parameters were automatically extr...

Fluorescence lifetime imaging microscopy (FLIM) can assess cell’s metabolism through the fluorescence of the co-enzymes NADH and FAD, which exhibit a double-exponential decay, with components related to free and protein-bound conditions. In vivo real time clinical imaging applications demand fast acquisition. As photodamage limits excitation power, this is best achieved using wide-field techniques, like time-gated FLIM, and algorithms that require few images to calculate the decay parameters. The rapid lifetime determination (RLD) algorithm requires only four images to analyze a double-exponential decay. Using computational simulations, we evaluated the accuracy and precision of RLD when measuring endogenous fluorescence lifetimes and metabolic free to protein-bound ratios, for total counts per pixel (TC) lower than 10 4 . The simulations were based on a time-gated FLIM instrument, accounting for its instrument response function, gain and noise. While the optimal acquisition setting depends on the values being measured, the accuracy of the free to protein-bound ratio α 2 /α 1 is stable for low gains and gate separations larger than 1000 ps, while its precision is almost constant for gate separations between 1500 and 2500 ps. For the gate separations and free to protein-bound ratios considered, the accuracy error can be as high as 30% and the precision error can reach 60%. Precision errors lower than 10% cannot be obtained. The best performance occurs for low camera gains and gate separations near 1800 ps. When considering the narrow physiological ranges for the free to protein-bound ratio, the precision errors can be confined to an interval between 10% and 20%. RLD is a valid option when for real time FLIM. The simulations and methodology presented here can be applied to any time-gated FLIM instrument and are useful to obtain the accuracy and precision limits for RLD in the demanding conditions of TC lower than 10 4 .

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