Corneal FLIM

Corneal diseases are the second major cause of blindness worldwide, according to the World Health Organization. Moreover, being a highly innervated tissue, corneal diseases cause severe pain and discomfort. So, non-invasive methods for early detection of corneal pathologies can have a large impact on the monitoring and management of patients and contribute to prevent disease progression to irreversible conditions.

The clinicians need an imaging tool capable of detecting the very early signs of cell dysfunction. In other words, a functional imaging modality with cell-level resolution could have a deep impact on understanding the mechanisms causing corneal diseases, on the detection of disease signs at very early stages and, ultimately, on the patients’ prognosis. Fluorescence lifetime metabolic imaging microscopy has the potential of fulfilling this need.

This research project comprised the development and evaluation of two different approaches for implementing fluorescence lifetime metabolic imaging microscopy, both having the cornea as final application:

 - Using multiphoton imaging techniques, namely two-photon excitation and second harmonic generation, with serial pixel imaging using laser scanning excitation and time-correlated single photon counting detection.

- Using one-photon excitation with parallel, wide-field imaging and time-gated detection through an intensified camera.

The first approach offered greater promise due to the inherent optical sectioning of multiphoton imaging, the use of safer infrared wavelengths and the possibility of exciting both NAD(P)H and FAD fluorescence with the same laser source. However, there were concerns on the safety of the high peak radiant powers required by multiphoton excitation and on the very high cost of lasers capable of emitting pulses with a few tenths of femtoseconds. 

The second approach, could use much less expensive picosecond diode lasers and was considered safe for in vivo ocular imaging, due to the results already obtained in time-resolved fluorescence imaging of the retina by the group of Prof. Dietrich Schweitzer (Schweitzer et al., 2004a). However, there were high concerns on the possibility of obtaining optical sectioning on a thick transparent tissue, on the suitability of Rapid Lifetime Determination algorithms for obtaining the decay parameters with adequate accuracy and precision and on obtaining an adequate signal using only FAD fluorescence. Relying on NAD(P)H fluorescence was not considered since one-photon excitation of this metabolic co-factor implies ultraviolet radiation that was deemed unsafe for in vivo ocular imaging, due to its phototoxicity.

Part of this project was funded by FCT:


IBILI - Institute for Biomedical Research on Light and Image

Instrumentation Center - University of Coimbra

Funding: 145.100 €

Funding entity: FCT – Fundação para a Ciência e Tecnologia

Reference: PTDC/SAU-ENB/122128/2010

Start: 1st of May, 2012

End: 31st of August, 2015

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I am a professor and a researcher at the University of Coimbra, Portugal.

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