Adaptive optics fluorescence lifetime imaging ophthalmoscopy of in vivo human retinal pigment epithelium

Abstract

The intrinsic fluorescence properties of lipofuscin – naturally occurring granules that accumulate in the retinal pigment epithelium – are a potential biomarker for the health of the eye. A new modality is described here which combines adaptive optics technology with fluorescence lifetime detection, allowing for the investigation of functional and compositional differences within the eye and between subjects. This new adaptive optics fluorescence lifetime imaging ophthalmoscope was demonstrated in 6 subjects. Repeated measurements between visits had a minimum intraclass correlation coefficient of 0.59 Although the light levels were well below maximum permissible exposures, the safety of the imaging paradigm was tested using clinical measures; no concerns were raised. This new technology allows for in vivo adaptive optics fluorescence lifetime imaging of the human RPE mosaic.

Lipofuscin, Its Origin, Properties, and Contribution to Retinal Fluorescence as a Potential Biomarker of Oxidative Damage to the Retina

“…This can be achieved by employing short pulses of high-intensity near-infrared laser. The most recent development in clinical imaging of fundus fluorescence is spectral imaging of fluorescence emitted in the range of 400-700 nm upon intense pulse excitation with two photons of near-infrared wavelengths and the use of adaptive optics [217,254,255,295,[308][309][310][311][312][313][314][315][316][317][318][319][320][321][322]. The emitted light can be spectrally analyzed, which enables the detection of specific fluorophores.…”

“…It appears that the recent developments in imaging techniques, which include combined OCT and fluorescence imaging of the fundus with subcellular resolution make such monitoring in vivo of the spectral changes in fluorescence emission of lipofuscin feasible [254,295,322]. The combined use of OCT and SLO allows for the identification of areas with SDD or drusen, which can also be a source of fluorescence.…”

“…The combined use of OCT and SLO allows for the identification of areas with SDD or drusen, which can also be a source of fluorescence. The subcellular resolution of fluorescence imaging is enabled by TPEF-SLO, which limits the area of the excitation, and imaging of both -fluorescence and reflectance [254,295,322]. The latter is used to identify the retinal layer where the laser light is focused so it is the origin of the fluorescence.…”

“…Moreover, due to excitation with near-infrared lasers, the artifacts introduced by the anterior segment of the eye are minimized. Importantly, it has been demonstrated that such imaging can be done safely for the human eye in vivo [254,295,322].…”

“…Boguslawski and colleagues have demonstrated that safe imaging of the human fundus in vivo by TPEF-SLO provides a sufficient signal-to-noise ratio for informative imaging of the reflectance and fluorescence emission when collected in distinct spectral windows obtained by bandpass filters: 400-600 nm, 594-646 nm, 500-540 nm, and 400-550 nm [254,295,322]. However, the excitation source used in this instrument providing 76 fs pulses of 780 nm wavelength excites multiple retinal fluorophores and photosensitizers, including not only lipofuscin but also retinoids, flavins, and porphyrins.…”