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.

Keywords: cornea; corneal collagen organization; fluorescence lifetime imaging; optical metabolic imaging; second-harmonic generation; two-photon imaging.

Figure 1

Representation of the human cornea cross-section. Reproduced from [7].

Figure 2

Jablonski diagrams of one-photon excitation (1PEF) and two-photon excitation (TPEF) fluorescence (a) and energy level diagram of second-harmonic generation (SHG) (b). The photon energy absorption by the molecule promotes its excitation from the ground state ($S_0)$ to a higher energy state ($S_1$), followed by internal conversion (IC) and relaxation by photon emission. In SHG, the energy of two photons with angular frequency $\omega$ is converted into a single photon with double the energy and angular frequency $2\omega$. $E$—energy; $E_{min}$—energy difference between $S_0$ and $S_1$; $E_2$—half of $E_{min}$; $E_F$—energy of fluorescence photons; $E_{SHG}$—energy of SHG photons; $h$—Planck’s constant; $\nu$—frequency; ${\nu }_F$—frequency of fluorescence photons; ${\nu }_{SHG}$—frequency of SHG photons ($=2\nu$).

Figure 3

Schematic representation of time-gated (a) and time-correlated single photon counting (b) principles of operation.

Figure 4

Corneal collagen hierarchic organization (a), and influence of fibril diameter and packing arrangement on the directionality and intensity of second-harmonic generation (SHG) signals (b). Arrows indicate the direction of signal propagation. $E$—energy; $SH{G}_F$ and $SH{G}_B$—forward- and backward-generated SHG; $L_{c}F$ and $L_{c}B$—coherence length of $SH{G}_F$ and $SH{G}_B$; ${\lambda }_{SHG}$—SHG wavelength; $\mathsf{\Delta }k$—phase mismatch.

Figure 5

Schematic representation of the optical setup of a two-photon imaging microscope with multichannel detection combined with a time-correlated single photon unit for fluorescence lifetime imaging. CFD—constant fraction discriminator; TAC—time-to-amplitude converter; ADC—analog-to-digital converter.

Figure 6

3D composite representation of the porcine cornea reconstructed from autofluorescence (green) and second-harmonic generation (red) images of the tissue (a), and depth-wise en-face images of the human cornea autofluorescence (green) and second-harmonic generation (red) (b). Images were acquired with the multiphoton tomograph MPTflex (JenLab, GmbH) using an excitation wavelength of 760 $\mathrm{nm}$. Scale bars = 20 $\mathsf{\mu }\mathrm{m}$.

Figure 7

Cross-sectional autofluorescence (AF) lifetime images color-coded for mean AF lifetime (as indicated in the color bar) of the keratoconus (KC) and keratoconus crosslinked (KC-CXL) corneas 24 h after treatment (a), corresponding mean AF lifetime decay distributions (b) and phasor plots (c). Images were acquired with the multiphoton tomograph MPTflex (JenLab, GmbH) using an excitation wavelength of 760 $\mathrm{nm}$. Fluorescence lifetime data analysis was performed using SPCImage vs 6.2 (Becker & Hickl GmbH, Berlin, Germany). Scale bars = 20 $\mathsf{\mu }\mathrm{m}$.