2PM includes both two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG). 2PEF is very similar to traditional fluorescence, except two photons of a lower energy are simultaneously absorbed to excite a fluorophore. The excited fluorophore subsequently fluoresces a single photon of the appropriate emission wavelength. 2PEF occurs with both endogenous and exogenous fluorophores. 2PEF can be used to excite endogenous biologic chromophores such as NAD(P)H, collagen, elastin, and melanin [13
]. Imaging by this method is often referred to as two-photon excitation autofluorescence (2PAF) or autofluorescence (AF) in short, since the fluorescence results from the intrinsic properties of these molecules and not from any external fluorescent label. Another nonlinear process that occurs with 2PM is second harmonic generation (SHG). SHG can only occur with non-centrosymmetric (asymmetric) macromolecular structures. Collagen fibers can simultaneously “scatter” two lower-energy photons as a single photon of twice the energy. SHG signal occurs at a distinct wavelength (half the excitation wavelength) and can be separated from tissue autofluorescence using a spectral detector.
1PM uses excitation wavelengths in the visible range (400–600 nm), which undergo significant optical scattering and material absorption in biologic samples. This limits 1PM visualization to within 100 µm of the surface of the tissue (reviewed in [16
]). 2PM is therefore much better suited to deep tissue imaging. For example, living skin has been imaged by 2PM to a depth of 350 µm by visualizing the AF of the skin’s extracellular matrix and melanin [17
]. The resolution in this study was determined to be 0.5–1 µm lateral by 3–5 µm axial, which is on par with typical a 5 µm thick histological section. Because of its great penetrating ability, 2PM has been used to successfully image the intact human cornea [18
] as well as flat-mounts of human retina and retinal pigment epithelium (RPE) [19
]. Furthermore, 2PM has been used to image the RPE and retina within the intact eye of rodents [21
]. A recent publication performed the first simultaneous 2PEF and third harmonic imaging on the cornea to detect elastin and collagen structures, and also showed the collagen and elastin structures of the TM [22
]. The authors were able to discern the prominent Schwalbe’s line, but they did not perform the deep-tissue imaging that is presented here, and also did not perform parallel imaging with a nuclear stain to simultaneously detect TM endothelial cells.
In this study, 2PM was used to image the native TM region of the human eye by AF and SHG. This method has the advantage over light microscopy of histological sections or EM on ultra-thin sections by being performed on unprocessed tissue. This eliminates distortions within the tissue due to infusion of fixatives, shrinkage of tissue due to alcohols, and changes to fine tissue morphology that can occur with heat-infusion of paraffin. There is presently no evidence that image artifacts are created from the hardware or software that we discuss in this report. In fact, since the software is calibrated to the optical properties of the objective lens, the software should correct for any distortions introduced by the imaging hardware. The TM was imaged en face by SHG and the ‘meshwork’ was found to have fluid spaces of non-uniform size, in confirmation of the organizational structures shown by quick-freeze deep etching EM of Gong et al. [10
]. Additionally, we were able to detect structures consistent with pores found in the IWSC (reviewed in [11
]). In contrast 2PM allows additional capabilities in comparison to EM by allowing fluorescent staining to highlight certain cell structures or proteins, and by allowing chemical-specific imaging by auto-fluorescence. The ability of z-resolution imaging to obtain full 3D structures of unprocessed samples is another key advantage.
The imaging presented here represents microscopy starting from the aqueous humor face of the TM toward the JTM region, with the microscope objective located within a millimeter of the surface of the tissue. We realize that this would not be amenable to the clinical imaging of patient TM, but we believe these 2PM images validate this method and represent a great leap forward in understanding the native structures within the TM. These findings have served as the foundation for performing current studies investigating 2PM imaging techniques in perfused whole human eye specimens using proprietary systems.
The hallmark indicator of glaucoma, elevated intraocular pressure (IOP), is believed to result from dysfunction in the anterior fluid drainage system of the eye. It is clear that surrogate metrics for glaucoma, such as IOP, are not fool-proof in diagnosing disease. Patients with elevated IOP often do not develop glaucoma and patients with glaucoma do not necessarily have high IOP [23
]. Other metrics are needed for the care of glaucoma patients to identify cellular dysfunction in vivo rather than using the surrogate metrics that have limited diagnostic sensitivity and specificity. This study shows that 2PM is useful for imaging tissues responsible for the regulation, or dysregulation, of aqueous humor outflow from the eye. Further studies are needed to explore the safety of implementing this imaging device in clinical practice, and to move forward with the creation of novel methods to bypass the limbal tissues that shield the drainage system from the reach of currently available 2PM imaging microscopes.