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

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.

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

“…Analogous to integral approximation methods in calculus, having more time gates with smaller widths results in higher lifetime accuracy, particularly for fluorophores with short lifetimes or multiexponential decay, while having a longer overall detection time improves accuracy for longer fluorescence lifetimes [ 46 ]. Adding more gates or increasing the detection window size leads to longer acquisition times and lower throughput; however, analysis algorithms [ 21 , 47 ] and even deep learning [ 22 ] have been implemented to maintain accuracy while collecting less images or gates. Similar detectors used for TCSPC can be used in time-gated FLIM, including modified SPAD arrays with gating electronics.…”

Can we use rapid lifetime determination for fast, fluorescence lifetime based, metabolic imaging? Precision and accuracy of double-exponential decay measurements with low total counts

“…The effect of the instrument’s IRF in the measurement accuracy can be corrected by deconvoluting the IRF from the acquired data. We already demonstrated the benefits of this approach on accuracy improvement for single-exponential decays [23]. The procedure is based in the iterative convolution of the measured IRF with synthetic fluorescence decay data, generated in a similar way to what we described in the “Fluorescence decay simulations” section.…”

“…Therefore, expensive femtosecond lasers are required when measuring sub-nanosecond fluorescence lifetimes. IRF deconvolution algorithms can be used to improve the accuracy of RLD, as we demonstrated recently for single-exponential decays, opening the possibility of using low cost pulsed diode laser sources to determine accurately fluorescence lifetimes in the sub-nanosecond range [23].…”

Applications of machine learning in time-domain fluorescence lifetime imaging: a review

“…E.g, the Fourier space approach of phasors [ 69 , 70 ]. Focusing on time-domain systems, two basic forms of time-determined fluorescence (aka TRF) can be described, namely time-gating [ 71 ] and TCSPC [ 72 ]. From a data analysis perspective, FLIm systems designed by the above techniques mainly differ in terms of temporal resolution and video frame rate [ 14 ].…”