Two-Photon Imaging for Non-Invasive Corneal Examination.

Two-photon imaging (TPI) microscopy, namely, two-photon excited fluorescence (TPEF), fluorescence lifetime imaging (FLIM), and second-harmonic generation (SHG) modalities, has emerged in the past years as a powerful tool for the examination of biological tissues. These modalities rely on different contrast mechanisms and are often used simultaneously to provide complementary information on morphology, metabolism, and structural properties of the imaged tissue. The cornea, being a transparent tissue, rich in collagen and with several cellular layers, is well-suited to be imaged by TPI microscopy. In this review, we discuss the physical principles behind TPI as well as its instrumentation. We also provide an overview of the current advances in TPI instrumentation and image analysis. We describe how TPI can be leveraged to retrieve unique information on the cornea and to complement the information provided by current clinical devices. The present state of corneal TPI is outlined. Finally, we discuss the obstacles that must be overcome and offer perspectives and outlooks to make clinical TPI of the human cornea a reality.

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.

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 104. 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 α2/α1 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 104.

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

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.