Two-Photon Imaging for Non-Invasive Corneal Examination

Abstract

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.

Two-photon excitation fluorescence in ophthalmology: safety and improved imaging for functional diagnostics

“…Currently the 2Ph absorption, after non-radiant energy relaxation, is explored in medical context in three main ways: (i) in the emission of a single photon in the visible range via fluorescence emission (TPEF), (ii) or in case of instantaneous process and no energy lost, in a second-harmonic generation (SHG) scattered UV light ( 7 , 10 ), and (iii) in fluorescence lifetime imaging microscopy (FLIM) ( Figures 1E-I ).…”

“…In TPEF, two less-energetic (i.e., near IR range) photons are used to excite molecules instead of a single high-energy photon (i.e., in the UV range), thus bypassing the usage of UV illumination limitations. This approach provides multiple advantages for using TPEF as a diagnostic tool, including: (i) it causes less damage ( 8 – 10 ), as it has a quadratic dependence on the excitation intensity (instead of the linear dependency of one-photon excitation); (ii) it has a more profound penetration power in tissues because the excitation is limited to the focal volume with high photon density ( 8 – 10 , 20 ) ( Figures 1E – F ), and there is decreased scattering ( 8 – 10 , 32 ); and (iii) in ophthalmology, it avoids the sequestration of UV light by the anterior segment of the human eye ( 39 , 40 ) ( Figure 1N ).…”

“…The cornea is the outermost anterior part of the eye, and it is transparent and avascular to allow light transmission and refraction ( 10 ). Its thickness varies from 500 μm in the center to 700 μm toward the periphery.…”

“…The cornea is comprised of five main layers (epithelium, Browman’s layer, stroma, Descemet’s membrane, and endothelium). The stroma accounts for ~90% of the corneal thickness, and is composed of stacked collagen lamellae ( 7 , 10 ). SHG allows the visualization of non-centrosymmetric molecules, such as collagen ( 7 , 41 , 42 ).…”

On the quantitative analysis of lamellar collagen arrangement with second-harmonic generation imaging

“…Collagen accounts for approximately 90% of corneal thickness and plays a key role in the shape, transparency, and optical properties of the cornea. Changes in collagen organization can severely affect vision and have been amply investigated by SHG [ 7 ]. The advantages of SHG were already demonstrated in the diagnosis of corneal diseases [ 8 – 11 ] as well as to evaluate the outcome of therapeutic procedures, such as corneal collagen crosslinking [ 12 – 16 ].…”