The eye is also very special for another reason. Our vision depends on vitamin A (all-
trans-retinol), a fluorescent isoprenoid that is a precursor for many other related chromophores.
21 All-
trans-retinol and its fatty acid esters are particularly useful for imaging because of their characteristic intrinsic fluorescence.
22 Retinol fluorescence is highly sensitive to solvents. Its quantum yield varies from 0.07 in cyclohexane,
23 0.027 in hexane,
24,25 0.016 in 3-methylpentane/triethylamine,
26 and 0.011 in ethanol
27 to 0.015 in isopropanol.
28 This sensitivity to the environment could be very useful when combined with analytical methods to distinguish different biochemical/cellular processes in the retina. Vitamin A is a conjugated polyene compound with several naturally and artificially generated isomers. These isomers are also fluorescent with quantum yields in 3-methylpentane-methylcyclo-hexane of 0.006 and 0.007 for 9- and 11-
cis isomers, respectively.
22 Interestingly, in contrast to alcohols, retinal aldehydes exhibit far less fluorescence.
29 Another important group of retinal chromophores are retinal condensation products, which are much more fluorescent than retinols, or any other metabolic product of retinoids.
30 During natural aging, condensation products such as di-retinoid-pyridinium-ethanolamine (A2E) and compounds of similar chemical nature and their oxidized derivatives accumulate within RPE contributing to increased fluorescence of this cell layer.
31,32 Fundus fluorescence of human eye increases significantly with age, has a broad emission between 500 and 750 nm,
33 and resembles A2E fluorescence of human RPE in culture.
34 At 488-nm excitation, Bruch's membrane and sub-RPE deposits in normal eyes exhibited enhanced fluorescence in eyes of donors with AMD.
35 Other all-trans-retinal conjugation products and A2E are considered as biomarkers for elevated levels of potentially toxic retinoids that accelerate the development of retinal degenerative diseases such as Stargardt disease and AMD.
36,37 In more recent studies, Schweitzer et al.
38 used time-resolved autofluorescence measurements recorded simultaneously in 16 spectral channels (445–605 nm) on fundus samples from a donor with significant extramacular drusen. These authors detected a bright fluorescence from RPE lipofuscin with a maximum at 510 nm and a lifetime of 385 ps. This fluorescence declined at longer wavelengths but was still substantial at 600 nm. These investigators also characterized different types of drusen
39 ; most that did not contain lipofuscin and exhibited fluorescence with a maximum at 470 nm and lifetime of 1785 ps. Other researchers have observed the absence of elevated levels of A2E in the macula.
31,40 It was also proposed that A2E and other precursors of lipofuscin have a photoreceptor-derived origin as a result of continuous transport of free 11-
cis-retinal to visual pigments.
41 However, recent imaging of retinal degeneration after an intense bleach in a Stargardt disease–mouse model clearly demonstrated that condensation products are formed in photoreceptors, but only as a result of rhodopsin bleaching and not 11-
cis-retinal transport.
20 These aberrant fluorophores, that exhibit different spectral characteristics than retinols, are also readily detectible by 2PO imaging with a new class of femtosecond pulsed lasers with an extended tuning range.
9 Both NADH and NADPH display fluorescent spectral properties similar to retinoids,
42,43 and can contribute to the fluorescence signal in native tissue.
11 Thus, vitamin A–derived retinoids and dinucleotides are natural fluorophores with absorption spectra that respond to multiphoton excitation, bypassing the filter-like properties of the cornea and lens.