14 March 2016 Time-gated FLIM microscope for corneal metabolic imaging
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Abstract
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..
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Susana F. Silva, Susana F. Silva, Ana Batista, Ana Batista, José Paulo Domingues, José Paulo Domingues, Maria João Quadrado, Maria João Quadrado, António Miguel Morgado, António Miguel Morgado, } "Time-gated FLIM microscope for corneal metabolic imaging", Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97122C (14 March 2016); doi: 10.1117/12.2214182; https://doi.org/10.1117/12.2214182
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