These results demonstrate that it is possible to image cellular structures in the living primate eye with two-photon fluorescence. The specific fluorophore(s) that were excited remain(s) to be determined. Fluorophores previously imaged in ex vivo
photoreceptors have included mitochondrial flavin adenine dinucleotide (FAD) [15
], reduced pyridine nucleotides involved in cellular metabolism (NADH and NADPH, collectively referred to as NAD(P)H) [6
], A2-PE [16
] and all-trans
]. Because A2-PE and all-trans
-retinol are products of the visual cycle that are predominately located in outer segments, substantial amounts would not be expected in cone inner segments. Fluorophores in cone inner segments can also include 11-cis
] or compounds yet to be identified.
Because of the low number of emitted fluorescent photons, it is not currently possible to experimentally identify in vivo
fluorophores using the FAOSLO. NAD(P)H and retinol have similar two-photon excitation cross-sections [19
]. At 730 nm, the wavelength used for fluorescence excitation, the two-photon cross-section of FAD is almost double that of NAD(P)H, but follows a similar action spectrum until it peaks again near 900 nm [20
]. Although the emission peaks [6
] are slightly separated, NAD(P)H, FAD and retinol are all easily detected by the broad emission collection in the FAOSLO. Given their spectral properties, emission from other fluorophores, such as A2-PE [16
] or lipofuscin [21
], is unlikely to be collected in significant quantities.
two-photon imaging was achieved without observable retinal damage as assessed from 1 day to a year later by fundus photography and fluorescein angiography. Moreover, the same location could be imaged on separate occasions with no detectable change in the appearance of the fluorescence or reflectance images. If this method were to be used in humans, the light levels required would have to comply with the current American National Standard for the Safe Use of Lasers [22
] which provides guidance for calculating maximum permissible exposures (MPEs) for ocular light incorporating about an order of magnitude of safety. MPE calculations for a scanning laser ophthalmoscope have been described in detail elsewhere [4
]. For the FAOSLO, with imaging conditions described herein, the MPE is 665 μW for a 12 minute exposure (the acquisition time for generating a single two-photon image). Successful in vivo
two-photon imaging was achieved by using 3 to 3.5 mW of excitation power incident on the cornea, over 4.5 times the 12 minute ANSI MPE, but less than the damage threshold expected from experimental data for continuous wave illumination [24
], a suitable comparison for thermal damage. With improvements in light efficiency, safe two-photon imaging of the living human eye appears to be feasible.
imaging can provide insight into the molecular changes that occur during the phototransduction cascade and subsequent visual pigment regeneration. Improved understanding of the processes of vision in living healthy and diseased retina can help advance therapies for successful aging, as well as improve the diagnosis and treatment of some retinal pathologies [2
]. Intrinsic signals have been investigated in vivo
at low spatial resolution with a fundus camera [25
] and at the cellular scale with OCT [27
], an AOSLO [28
] and an AO flood-illuminated camera [29
]. In response to visible light stimuli, positive and negative changes in near-infrared reflectance have been observed, the origins of which are not completely understood. The observed intrinsic signals have been attributed to changes in vascular blood flow [25
] and/or photoreceptor changes such as, membrane hyperpolarisation [26
], cell swelling [26
], and changes in refractive index or scattering properties [29
]. These are indirect measures of the effects of chemical changes occurring in response to photoreceptor activation. Two-photon imaging of the living eye at a cellular scale has the potential to directly measure the changes in concentrations of fluorescent molecules involved in visual excitation. Using two-photon imaging in an isolated frog rod, Chen and colleagues [6
] demonstrated an increase in the two-photon excited fluorescence signal following exposure to visible light. Spectroscopic measurements identified the fluorophores as NAD(P)H in the rod inner segments and all-trans
-retinol in the outer segments. They also obtained similar results with isolated mouse retina. Here, we have shown, in vivo
and in an ex vivo
preparation, an increase in two-photon fluorescence signal from cone inner segments in response to light exposures that would result in cone bleaching.
When enabled by a high-resolution adaptive optics scanning laser ophthalmoscope, two-photon imaging provides functional measurements at the cellular scale in the living eye. The nature of current functional measurements is still under investigation. The increased fluorescence may represent the creation of new fluorescent molecules or an increase in the concentration of existing fluorescent molecules either directly by the two-photon excitation light or by its stimulation of a visual response. The capability to image changes in cellular metabolism (if imaging FAD or NAD(P)H) or the influx and conversion of 11-cis
-retinol in cone inner segments in response to visual stimuli is of interest, not only in young and aging healthy eyes, but also in eyes with retinal pathology. If the two-photon imaging signal emanates from NAD(P)H or FAD, mitochondrial dysfunction, such as that originating from Leber’s hereditary optic neuropathy [30
] or NARP (neurogenic muscle weakness, ataxia, retinitis pigmentosa) [31
], the technique can display different fluorescence changes over time when compared to normal healthy eyes. Altered responses to light may also be observed with diseases of the visual cycle, including Leber’s congenital amaurosis and Stargardt disease [2
]. Two-photon fluorescence imaging capabilities could be highly useful for monitoring the efficacy of proposed therapies.