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Journal of Healthcare Engineering
Volume 2018, Article ID 1371386, 7 pages
https://doi.org/10.1155/2018/1371386
Research Article

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

1Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal
2Department of Physics, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

Correspondence should be addressed to António Miguel Morgado; tp.cu@odagromleugim

Received 26 August 2017; Revised 21 November 2017; Accepted 3 December 2017; Published 16 January 2018

Academic Editor: Alireza Vard

Copyright © 2018 Susana F. Silva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.