1.

Introduction

Vertebrate retinas share a common inverted structure, i.e., the photoreceptor layer is located at the far-most position in respect to incident light. Although the inverted arrangement seems unfavorable at first sight (the light has to travel through the whole neurosensory retina to reach photoreceptors), it is vital for the retina to survive in an environment with high photon and oxygen flux. The opaque layers, i.e., the retinal pigment epithelium (RPE) and the choroid, are indispensable to maintain the normal metabolism and visual function of the retina.^{1, 2} RPE cells are highly specialized cells that serve as nurse cells for the retina with multiple essential functions.^{1, 2} First, RPE digests the shed photoreceptor outer segments on a daily basis, transforms the photoisomerized all-trans-retinal back into 11-cis-retinal, and maintains the excitability of photoreceptors. Second, RPE takes up and delivers nutrients to the neurosensory retina and transports the metabolic end products to the choroid. Third, RPE has elaborate mechanisms to remove the toxic molecules and free radicals produced by the light, contributing to a stable and optimum retinal environment. Fourth, the melanin pigment in the RPE protects the photoreceptors from short-wavelength light damage and shields scattered light from the sclera. The intensive metabolic activities in the RPE require a good blood supply, which is provided by the choroid being in intimate contact with the RPE. The choroid is the layer that actively transports the metabolic waste and nutrients from and to the photoreceptors, respectively.³ The choroid takes approximately $85\%$ of the ocular blood flow and is remarkable for its high blood flow rate. Besides its transport function, the choriocapillaries act as a heat sink to reduce subretinal temperature and photochemical injury to the RPE.⁴ A failure of the functions of either RPE or choroid may lead to degeneration of photoreceptors, impairment in visual acuity, and eventually to blindness.

Age-related macular degeneration (AMD) is the principal cause of irreversible loss of vision and registered legal blindness for aging people in developed countries. To place this in perspective, $35\%$ of the human population older than 75 years have some degree of AMD.^{5, 6} AMD is characterized by the progressive degeneration of photoreceptors, RPE, and choroiod.^{7, 8} The earliest visible abnormality in AMD is the extracellular accumulation of drusen (waste material) underneath the RPE cell monolayer. Despite extensive research, the trigger events and the primary injury sites of AMD have always been subjects of considerable debate. Fundamentally, there are two hypotheses regarding the primary cause of AMD.⁵ One claims a vascular origin associated with an imbalance between angiogenetic and antiangiogenetic factors. The second suggests that dysfunction of the RPE is responsible for AMD. The development of AMD is thought to result from an accentuation of the aging process, since most of the structural abnormalities presented in AMD are also observed in normal aged eyes. A noninvasive, high resolution, and large sensing depth imaging modality is vital to delineate the age-related structural abnormalities in the fragile, pigmented retina-choroid complex and to provide clues to the events that transform normal aging processes into early AMD developments.

Although for most imaging applications autofluorescence is a source of background and may need to be suppressed, autofluorescence is particularly valuable in ophthalmology and has been successfully used for diagnostic in-vivo autofluorescence imaging as well as for experimental ex-vivo imaging techniques. ^{9, 10, 11, 12, 13, 14} As illustrated in Fig. 1 , the human retina appears to be an ideal target for endogenous fluorescence imaging. The major autofluorescent sources in human retina include all-trans-retinol and NAD(P)H in photoreceptors, lipofuscin and melanin in the RPE, elastin in Bruch’s membrane, and red/white blood cells in choroid. All of them are among the most vital components in the retina-choroid complex. However, the most effective excitation light for the retinal autofluorescence is in the blue-UV range, which is either strongly scattered or absorbed in the opaque RPE-choroid complex and may induce severe photodamage. The advantages of autofluorescence retinal imaging cannot be appreciated with conventional single-photon imaging. The advent of ultra-fast pulsed laser sources allowed the use of a nonlinear absorption process for two-photon microscopic fluorescence imaging.^{15, 16} Near-infrared (NIR) ultra-fast lasers are employed as the excitation light source, resulting in largely increased sensing depth and reduced photodamage. ^{17, 18, 19, 20, 21, 22} As a second-order effect, two-photon absorption is confined within the central focus of the illuminating beam. Lateral and depth discrimination are achievable without extra confocal pinholes,²³ permitting 3-D optical sectioning of thick retina-choroid complex. Although two-photon excited fluorescence (TPEF) imaging has been extensively applied in neurophysiology, developmental biology, and biopsy, ^{19, 20, 21, 22, 23, 24, 25} TPEF ophthalmic imaging is still in the early development stage. ^{25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36} To the best of our knowledge, we are the first to employ two-photon excited autofluorescence imaging to resolve the age-related structural abnormalities in the human retina-choroid complex prior to apparent pathological manifestations of AMD. As a simple and efficient method, TPEF imaging has tremendous potential in delineating the cellular structures of retina-choroid complex, and may provide fresh insights into the pathogenesis of blinding retinal diseases.

Fig. 1

Endogenous chromophores in the human photoreceptor-RPE-choroid complex. The major fluorophore in the outer segment of the photorecetor is all-trans-retinol. NAD(P)H is located in the photoreceptor inner segment and in the outer nuclear layer. Lipofuscin and melanin are the predominant fluorophores in the RPE. The Bruch’s membrane and choriocapillaries can be delineated based on the autofluorescence of elastin, and red/white blood cells.

2.

Materials and Methods

2.2.

Two-Photon Excited Autofluorescence Imaging

In this study, TPEF imaging was performed on a customized upright laser scanning microscope (Zeiss LSM 510 NLO, Zeiss, Jena, Germany) equipped with a femtosecond Ti:sapphire laser (Coherent Chameleon XR, Coherent Incorporated, Santa Clara, California). The wavelength of the $90\text{-}\mathrm{MHz}$ , $150\mathrm{fs}$ Ti:sapphire laser pulses is tunable from 705 to $980\mathrm{nm}$ . A $5\text{-}\mathrm{mm}\text{-}\mathrm{diam}$ retina/sclera tissue probe in the macular region was manually prepared with a surgical trephine. First, the neurosensory retina then the RPE were carefully removed from the tissue probe, allowing high resolution imaging of photoreceptors, RPE, and choroid, respectively. All prepared tissue specimens were transferred to a custom-made tissue dish filled with PBS (pH 7.4) solution and imaged with a large working distance water-immersion objective (Zeiss, $63\times ,\mathrm{NA}$ 1.0). The average laser power at the back focal plane of the objective was attenuated below $\sim 4\mathrm{mW}$ for RPE imaging and was kept between $4\mathrm{mW}\sim 10\mathrm{mW}$ for 3D photoreceptor and choroid imaging, which are close to the previously reported damage thresholds.^{35, 36} We did not observe noticeable photodamages to the fixed retina during imaging. Through a set of dichroic beamsplitters, the autofluorescence signals were detected by photomultiplier tubes assigned to different spectral windows, namely, 500 to $550\mathrm{nm}$ for photoreceptor imaging (Fig. 2 ), 435 to 485 and 515 to $700\mathrm{nm}$ for RPE-choroid-Bruch’s membrane imaging [Figs. 3, 4b, 5 ], and 500 to 550 and 575 to $640\mathrm{nm}$ for lipofuscin and melanin imaging [Fig. 4a]. The wavelength of the Ti:sapphire laser for TPEF imaging was set to $800\mathrm{nm}$ , except for Fig. 4a $\left(916\mathrm{nm}\right)$ . The cross section images of photoreceptors in Figs. 2c and 2d, and Figs. 2g and 2h were reconstructed from $70\text{-}\mathrm{\mu }\mathrm{m}$ -depth image stacks with slice intervals of $1\mathrm{\mu }\mathrm{m}$ .

Fig. 2

Human cone and rod photoreceptor mosaic delineated by TPEF imaging. (a) and (b) Cone and rod mosaic in fovea, parafovea ( $1.2\text{-}\mathrm{mm}$ eccentricity) of the 19-year-old eye. (c) and (d) Cross section images of foveal, parafoveal photoreceptors of the 19-year-old eye. The locations of the optical slices of (a) and (b) are indicated by the arrowheads in (c) and (d). (e) through (h) Corresponding single slice and cross section images of the 57-year-old photoreceptors in fovea and parafovea ( $1.2\text{-}\mathrm{mm}$ eccentricity), respectively. ONL is the photoreceptor outer nuclear layer; IS is the inner segments; and OS is the outer segments. Scale bar, $10\mathrm{\mu }\mathrm{m}$ .

Fig. 3

Morphometric and spectral characterizations of single lipofuscin granules in human RPE cells. TPEF signals from the 19-year-old macular RPE cells were acquired with two spectral windows color coded in (a) green $(435<{\lambda }_{AF}<485\mathrm{nm})$ and (b) red $(515<{\lambda }_{AF}<700\mathrm{nm})$ . (c) Merged TPEF images of the 19-year-old RPE cells. (d), (e), and (f) Corresponding TPEF images of the 64-year-old macular RPE cells (green channel and red channel, merged, color online only), respectively. Large lipofuscin granules in the 64-year-old eye demonstrate significantly blue-shifted autofluorescence. Scale bar, $10\mathrm{\mu }\mathrm{m}$ .

Fig. 4

(a) Interdependence of melanin and lipofuscin pigment granules in the RPE. Most of the melanin pigment granules (as indicated by the arrows) are located in the apical pole of the RPE cells. In the RPE cells (55 year old), which contain enlarged lipofuscin granules with blue-shifted autofluorescence, the amount of melanin granules decreased remarkably. (b) Blue-light irradiation elicits autofluorescence shift of lipofuscin granules in the RPE (64 year old). Compared with the normal RPE cells on the left, the blue-light-illuminated RPE cells on the right demonstrate significantly blue-shifted autofluorescence. Scale bar, $10\mathrm{\mu }\mathrm{m}$ .

Fig. 5

Age-related choroidal structural and circulation alterations. Bruch’s membrane [(a) and (d)] and choriocapillaries [(b) and (e)] in the 19- and 64-year-old choroids, respectively. Black asterisks: jammed red blood cells. White arrowheads: representative white blood cells (neutrophils). (c) and (f) The choroidal layer immediately underneath the choriocapillaries. The interwoven fibrous structures are present in (f) the 64-year-old choroid, but not in (c) the 19-year-old choroid. Scale bar, $10\mathrm{\mu }\mathrm{m}$ .

Table 1

Age-related structural abnormalities in the human retina-choroid complex.

Retina-choroid complex	Endogenous fluorophores	Age-related structural abnormalities
Photoreceptors	NAD(P)H All-trans-retinol	Autofluorescent intracellular inclusions in elderly cones. More variable outer segments with increasing age.
RPE	A2E Melanosome	Excessive accumulation of lipofuscin. Blue-shifted fluorescence from enlarged lipofuscin granules. Melanin depletion in RPE cells with blue-shifted fluorescence.
Bruch’s membrane	Elastin Extracellular deposit	Significantly increased autofluorescence. Fibrillar structures in Bruch’s membrane.
Choroid	Elastin White/red blood cells	Probable decrease in choroidal circulation. Fibrillar structures beneath choriocapillaries.

3.

Results

4.

Discussion

Based on the endogenous chromophores in the human retina-choroid complex, the age-related structural abnormalities (Table 1 ) in the photoreceptors, RPE cells, Bruch’s membrane, and the choroid were successfully delineated with two-photon excited autofluorescence imaging ex vivo. Individual cones or rods were resolved based on the autofluorescence from all-trans-retinol and NAD(P)H in the photoreceptor outer and inner segments, respectively. As age increases, the average densities of rods and cones appear unchanged in both fovea and parafovea. Our observations are inconsistent with the previous morphometric studies, where the parafoveal rod loss is generally greater than foveal cone loss during aging.^{55, 56} The discrepancy may result from the fact that all of the donor eyes under our investigations have normal vision and are younger than 64 years. Strongly autofluorescent intracellular inclusions were found in the aged cones. The origin of these autofluorescent inclusions and their influence to the visual function of the cones are worth further investigations. Derived from incompletely digested photoreceptor outer segments, lipofuscin accumulates in the RPE and exhibits the strongest autofluorescence in the retina. In the aged RPE, the amount of lipofuscin granules increases significantly. The average size of the lipofuscin granules remains more or less unchanged. However, individual RPE cells containing enlarged lipofuscin granules with strongly blue-shifted autofluorescence were observed in the elderly RPE, which was accompanied by a significant reduction in the melanin content. In addition to its light absorption function, melanin may act as an antioxidant to protect the RPE from photo-oxidative stress.⁵⁷ The occurrence of blue-shifted lipofuscin autofluorescence coincides with the depletion of melanin pigments in the aged RPE, suggesting a complicated interdependence between lipofuscin and melanin in the RPE. In the choroid, autofluorescence from elastin and from red/white blood cells offers an unique opportunity to delineate Bruch’s membrane, choriocapillaries, and intercapillary pillars. The elderly Bruch’s membrane demonstrated a significant increase of autofluorescence, which may represent age-related accumulation of autofluorescent sub-RPE deposits.¹⁴ In the choroidal layer beneath Bruch’s membrane, an extraordinary high density of choriocapillaries was resolved, which appears essential for the choroid to sustain a high blood circulation rate. A remarkably high white-to-red blood cell ratio was observed in the postmortem choriocapillaries. Such a high white blood cell concentration may be advantageous for efficient digestion of the metabolic end products from retina through phagocytosis; however, whether it represents the status of the living eye needs to be verified through further in-vivo examinations. Previous studies have reported an age-related decrease in choroidal circulation,^{53, 54} which is consistent with our ex-vivo observations that red blood cells tend to jam in the elderly choriocapillaries. In the fovea, the choroidal circulation is the only source of nourishment to the retina and is responsible for removal of metabolites from the fovea. The ability of the choroid to sustain a healthy environment in the fovea strongly depends on an ample choroidal blood flow. A decrease in the choroidal flow rate may have deleterious effects to the retina. Besides the choroidal circulation alterations, the presence of prominent fibrillar structures in Bruch’s membrane and underneath the choriocapillaries is one of the most distinct characteristics of the elderly choroid. Such abnormalities in the extracellular matrix, plus age-related decrease in the choroidal blood flow, may impair the transport efficiency of the choriocapillaries, reduce the permeability of Bruch’s membrane, and give rise to further RPE and photoreceptor damages.

As an elegant and highly efficient method, TPEF imaging is especially valuable to delineate the thick, fragile, and opaque human retina-choroid complex ex vivo. The most vital components (photoreceptors, RPE, choriocapillaries, Bruch’s membrane) in the human retina-choroid complex can be examined with subcellular resolution. Unlike conventional histological and electron micrographic methods,^{58, 59} complicated and invasive procedures including labeling and slicing are unnecessary. Compared with other noninvasive methodologies like confocal imaging, TPEF is more appropriate to probe the opaque RPE-choroid complex, because the short-wavelength light, which is typically employed for single-photon fluorescence imaging, will be strongly absorbed in cornea, RPE, and choroid. Furthermore, based on the transparency of the eye to the NIR light, cellular events in the living retina may be unraveled by TPEF imaging. As demonstrated by an excellent recent study, TPEF imaging has huge potential in in-vivo imaging of the intact retina.²⁸ Better retina-choroid imaging modalities will accelerate progress in understanding the pathogenesis of AMD, and will help to design effective therapies for AMD at early stages of the disease. Without sacrificing the animals under investigation,⁵⁹ trigger events for retinal diseases and retinal degenerations, as well as possible regeneration processes under pharmaceutical interventions, may be monitored at the cellular level, and truly in vivo.⁶⁰