Abstract
purpose. To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon–excited fluorescence (TPEF) and confocal laser scanning microscopy.
methods. Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser.
results. TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 μm, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules.
conclusions. TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.
With the advent of confocal scanning laser ophthalmoscopy, it has become possible to visualize fundus autofluorescence (FAF) and its spatial distribution in vivo.
1 2 3 4 5 This method represents a tool to evaluate the RPE during ageing and in retinal diseases.
6 As shown by spectrometric investigations by Delori et al.
7 the FAF signal is mainly derived from lipofuscin (LF) granules in the RPE cell monolayer. Excessive accumulation of LF occurs as a result of lifelong phagocytosis of photoreceptor outer segments by postmitotic RPE cells and represents a common pathogenetic pathway in various monogenetic and complex retinal diseases including age-related macular degeneration (AMD).
8 9 10 11 12 Although AMD has become the most common cause in developed countries of registered blindness, its pathogenesis is still incompletely understood.
13 14
Once formed, the RPE cell apparently has no means either to degrade or release LF into the extracellular space. LF has been shown to contain toxic compounds including the dominant fluorophore A2-E, a pyridinium bisretinoid, and various other modified macromolecules. These compounds can interfere with normal cell function, and various molecular mechanisms of adverse effects have been elucidated recently.
15 16 17 18 19 20 Several lines of evidence suggest that oxidative damage plays an important role in lipofuscinogenesis, with antioxidant deficiency or pro-oxidant conditions being of relevance.
21 22 23 24
Physiological and disease-associated RPE cell morphology has been addressed by applying conventional microscopic methods, and typical features in relation to age and fundus localization have been described. RPE cell density decreases from the fovea to the outer peripheral fundus regions, and the total number of cells decreases by approximately 0.3% per year.
25 26 27
The advent of femtosecond laser sources led to the experimental implementation of two-photon–excited fluorescence (TPEF) microscopes.
28 Compared with conventional confocal fluorescence microscopes, two-photon absorption is confined to the region of peak intensity within the focus of the illuminating laser beam. An illustration of the localization of excitation by two-photon excitation has been published by Zipfel et al.
29 Diffraction-limited resolution and depth discrimination are achievable without extra pinholes, permitting high-resolution three-dimensional optical sectioning of thick tissues with reduced bleaching or phototoxic effects outside the laser focus. In contrast to the UV or blue-light excitation for one-photon–excited fluorescence (1PEF), infrared light is used for multiphoton excitation, which promises deeper sensing depth and less photodamage effects.
28 29 30 Owing to the rich resource of fluorescent dyes available for selective and nontoxic staining and innovative recording techniques such as fluorescence resonant energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP), multiphoton-excited fluorescence microscopy is widely applied in imaging living cells, probing single molecules, and investigating biochemical processes under physiological conditions. Compared with confocal microscopy, multiphoton microscopy is more suitable for imaging of living or photosensitive tissue, such as the human retina or RPE cells. Recently, the fine structure of the collagen fibrils in the cornea and sclera has been successfully resolved with second-harmonic imaging.
31 32 33 To the best of our knowledge, there has been no report of multiphoton microscopic imaging of human RPE cells. In this report, we introduce this novel experimental imaging method for visualizing morphologic and spectral characteristics of human RPE cells ex vivo as an initial step toward the implementation of a two-photon confocal scanning laser ophthalmoscope for in vivo investigations.
Four human eyecups from deceased white donors were used to prepare specimens for microscopic evaluation. The donor’s ages were 19, 55, 57, and 80 years. After removal of the anterior segment for cornea transplantation, the eyes were immediately fixed in 4% paraformaldehyde solution. There were no macroscopic visible retinal alterations such as soft drusen, retinal hemorrhage, or choroidal neovascularization. The anterior segment and vitreous were carefully removed. Specimens were trephined from the macular and peripheral areas with a 5-mm trephine. After removal of the neurosensory retina and the sclera, the RPE-Bruch’s membrane–choroid specimens were placed on a microscope slide and covered with a coverslip, with PBS buffer used to create a wet chamber.
Specimens were analyzed with a confocal laser scanning microscope (TCS SP2; Leica Microsystems, Bensheim, Germany) and a modified laser scanning multiphoton microscope (LSM 510 NLO; Carl Zeiss Meditec, Jena, Germany). The latter was equipped with a mode-locked femtosecond Ti:sapphire laser (Chameleon XR; Coherent Inc., Santa Clara, CA). 1PEF confocal imaging can be accomplished with the same microscope, with a CW Argon ion laser (λ = 458, 488, and 514 nm) used as an excitation source. The wavelength of the Ti:sapphire laser was tuneable from 720 to 980 nm. For TPEF imaging, the laser wavelength was set to 800 nm, which is equivalent to 400 nm (UV) excitation for 1PEF. The Ti:sapphire laser produces a 150-fs pulse with a repetition rate of 90 MHz. The laser output power was attenuated to 4 mW by an acoustic optic modulator (AOM; Carl Zeiss Meditec). This value of 4 mW is the average power before the objective. Because of the attenuation by the objective, the effective excitation power is less than 4 mW, which is close to the previous reported damage thresholds for TPEF imaging of Chinese hamster ovary cells or bovine adrenal chromaffin cells.
34 35 We did not observe noticeable photodamage to the fixed RPE cells at this power level. A 63×, high-numerical-aperture (NA = 1.2), water-immersion objective (Carl Zeiss Meditec) was used for TPEF imaging (lateral resolution, roughly 200 nm). The autofluorescence signals from RPE cells were collected with the same objective. Following a set of dichroic mirrors and beamsplitters, the autofluorescence signals were split into two color-coded detecting channels (non-descanned detection; green channel: 500<λ<550 nm, red channel: 575 <λ< 640 nm) and were detected by two photomultiplier tube (PMT) detectors, respectively. Imaging overlay of both channels and processing was performed by the operation software for the microscope to visualize the different autofluorescence of both channels in one image. The confocal laser scanning microscope (TCS SP2; Leica) was used to perform XYλ scanning. The complete emission spectrum of the autofluorescence from RPE cells in the range of 430 to 700 nm was measured with 8-nm accuracy through a motor-driven slit in front of the PMT detector. For both 1PEF and TPEF imaging, the acquisition time of a single 512 × 512-pixel image was generally within a few seconds, with galvanometer scanners. All the images presented in this article are single optical sections.
Human donor eyes were obtained from the eye bank of the Department of Ophthalmology, University of Bonn, Germany, and from the eye bank of the Department of Ophthalmology, University of Munich LMU, Germany. Informed consent for corneal transplantation and further use of tissue for research was obtained by a relative or documented in an organ donor pass in accordance with German law for organ donation.
In this study, TPEF imaging with a femtosecond Ti:sapphire laser allowed for investigation of morphologic and spectral characteristics of human RPE cells. The autofluorescence signal both ex vivo and in vivo mainly derived from fluorophores in LF granules.
6 7 Excessive LF accumulation in postmitotic RPE represents a common downstream pathogenetic pathway in various monogenetic and complex, multifactorial retinal diseases, including Stargardt’s disease and AMD. Therefore, it is of interest to record variations in LF load in RPE cells to gain a better understanding of associations with concomitant disease processes such as the development of geographic atrophy associated with severe visual loss. We have previously shown, using FAF imaging in advanced AMD that excessive LF accumulation precedes the enlargement of preexisting geographic atrophy as well as the development of new atrophic patches.
37 Before atrophy occurs, areas with excessive LF accumulation (i.e., increased FAF signal) are associated with corresponding impaired retinal sensitivity.
38 The resolution of other currently available imaging methods applied clinically including fundus photography, fluorescence angiography, optical coherence tomography, or ultrasonography neither allows for delineation of individual RPE cells nor for recording of intracellular metabolic changes, such as LF accumulation in the lysosomal compartment.
Recent biochemical analyses of isolated human LF granules revealed the presence of various compounds with toxic properties, including A2-E, which interferes with normal lysosomal function by inhibition of lysosomal degradation mediated by inhibition of the ATP-dependent lysosomal proton pump as well as by phototoxic and detergent properties.
15 16 17 18 19 20 In proteome analyses we have shown that a broad spectrum of LF proteins display specific posttranslational modifications, including malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGE), which again are thought to interfere with normal cell functions.
19
In the present study, the intracellular distribution of individual autofluorescent LF granules could readily be visualized with high-resolution TPEF imaging. LF granules were preferentially localized in the peripheral cytoplasm close to the cell borders when only low LF granule density was present. In RPE cells with higher LF granule density the granules also occupy central portions of the cell cytoplasm. Previously, a more basal location of LF granules has been described in young eyes in contrast to diffuse spread with age.
39 40 The particular distribution of intracellular LF granules allows for accurate delineation of the nonautofluorescent cell borders between neighboring RPE cells.
The ex vivo findings using TPEF are in accordance with previous reports of the use of other imaging methods. Herein, we have shown that TPEF imaging allows for visualization of human RPE cells and LF ex vivo. Besides its major advantages of large sensing depth and reduced photodamage, TPEF imaging can also be used to determine the autofluorescence spectrum of autofluorescent material in RPE cells. The broad, tunable excitation source (Ti:sapphire laser, λ = 720–980 nm) for TPEF imaging is equivalent to the UV and blue excitation range (λ = 360–490 nm). Therefore, the complete spectrum of the autofluorescence can be investigated with the femtosecond infrared beam. Through TPEF imaging of RPE cells, the morphology of the individual RPE cells and the distribution of the LF granules inside the RPE cells were visualized with a lateral resolution of 0.2 μm and an axial resolution of 1 μm. With 4 mW preobjective excitation power, TPEF did not induce any undesired photobleaching or photodamage, whereas previous studies involving TPEF microscopy have reported damage at this power level, imaging different cell lines and with other experimental setups including different pixel dwell time, scan time, numerical aperture of objective, and pulse-length.
34 35 41 The complexity of the damage mechanisms does not allow a general conclusion, but taking into account the presence of phototoxic photograph sensitizers and highly absorbing melanin in RPE, a further reduced average laser power or laser repetition rate may be necessary for TPEF characterization of living RPE cells.
The typical diameter of the LF granules was found to be below 1 μm, but a few were larger than 2 μm. Remarkably, enhanced blue-green fluorescence was observed from these larger LF granules. This is, to the best of our knowledge, the first description of fluorescence spectra of individual LF granules ex vivo. As a fingerprint of LF on the molecular level, blue shifted autofluorescence from LF granules may imply the presence of a different composition that may derive from other proteins or fluorophores inside the LF granules. However, the phenomenon may be related to thus far unknown byproducts in the metabolic process of individual RPE cells.
In summary, TPEF imaging is a novel tool suitable for visualizing autofluorescent LF granules in human RPE cells with submicrometer resolution. Compared with conventional 1PEF, TPEF with a tunable laser source allows for reduced photodamage and deeper sensing depth. This noninvasive technique can be used to assess further the pathophysiological role of dynamic age- and disease-associated alterations in cytoplasmic LF granule accumulation and distribution. It may also help to interpret in vivo fundus autofluorescence images recorded with confocal scanning laser ophthalmoscopy. Based on the proof of principle ex vivo in the present study, further work is now planned to evaluate of the safety of TPEF RPE imaging in RPE cultures and to develop a two-photon laser scanning ophthalmoscope for application in the animal model.
Supported by the German Research Council DFG (Deutsche Forschungsgemeinschaft), Bonn, Germany, Grant HO 1926/1-3, Priority Research Program Age-Related Macular Degeneration Grant SPP 1088, European Commission (EU) Grant FP6, and Integrated Project “EVI-GENORET” Grant LSHG-CT-2005-512036.
Submitted for publication December 8, 2005; revised April 27 and May 31, 2006; accepted August 14, 2006.
Disclosure:
A. Bindewald-Wittich, None;
M. Han, None;
S. Schmitz-Valckenberg, None;
S.R. Snyder, None;
G. Giese, None;
J. F. Bille, None;
F.G. Holz, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Frank G. Holz, Department of Ophthalmology, University of Bonn, Ernst-Abbe-Str. 2, D-53127 Bonn, Germany;
frank.holz@ukb.uni-bonn.de.
The authors are indebted to Ulrich Welge-Lüssen from the Department of Ophthalmology, University of Munich, Germany, for providing tissue samples.
von RückmannAV, FitzkeFW, BirdAC. Fundus autofluorescence in age-related macular disease imaged with a laser scanning ophthalmoscope. Invest Ophthalmol Vis Sci. 1997;38:478–486.
[PubMed]BellmannC, HolzFG, SchappO, et al. Topography of fundus autofluorescence with a new confocal scanning laser ophthalmoscope. [in German]Ophthalmologe. 1997;94:385–391.
[CrossRef] [PubMed]HolzFG, BellmannC, MargaritidisM, et al. Patterns of increased in vivo fundus autofluorescence in the junctional zone of geographic atrophy of the retinal pigment epithelium associated with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol. 1999;237:145–152.
[CrossRef] [PubMed]BindewaldA, JorzikJJ, LoeschA, SchuttF, HolzFG. Visualisation of retinal pigment epithelial (RPE) cells in vivo using digital high resolution confocal scanning laser ophthalmoscopy. Am J Ophthalmol. 2004;137:556–558.
[CrossRef] [PubMed]BindewaldA, JorzikJJ, RothF, HolzFG. cSLO digital fundus autofluorescence imaging. [in German]Ophthalmologe. 2005;102:259–264.
[CrossRef] [PubMed]DeloriFC, FlecknerMR, GogerDG, et al. Autofluorescence distribution associated with drusen in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2000;41:496–504.
[PubMed]DeloriFC, DoreyCK, StaurenghiG, ArendO, GogerDG, WeiterJJ. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci. 1995;36:718–729.
[PubMed]HoganMJ. Role of the retinal pigment epithelium in macular disease. Trans Am Acad Ophthalmol Otolaryngol. 1972;76:64–80.
[PubMed]SarksSH. Aging and degeneration in the macular region: a clinicopathological study. Br J Ophthalmol. 1976;60:324–341.
[CrossRef] [PubMed]DoreyCK, WuG, EbensteinD, et al. Cell loss in the aging retina. Relationship to lipofuscin accumulation and macular degeneration. Invest Ophthalmol Vis Sci. 1989;30:1691–1699.
[PubMed]BoultonME. Ageing of the retinal pigment epithelium.OsbornNN ChaderGJ eds. Retinal Research. 1991;126–147.Pergamon Press Oxford, UK.
BoultonM, Dayhaw-BarkerP. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye. 2001;15:384–389.
[CrossRef] [PubMed]BirdAC. Age related macular disease. Br J Ophthalmol. 96;80:1–2.
[CrossRef] HolzFG, PauleikhoffD, SpaideRF, BirdAC. Age-Related Macular Degeneration. 2004;Springer, Berlin, Heidelberg New York.
EldredGE. Age pigment structure. Nature. 1993;364:396.
HolzFG, SchuttF, KopitzJ, et al. Inhibition of lysosomal degradative functions in RPE cells by a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci. 1999;40:737–743.
[PubMed]SchuttF, DaviesS, KopitzJ, HolzFG, BoultonME. Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci. 2001;41:2303–2308.
SchuttF, BergmannM, HolzFG, KopitzJ. Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes. Graefes Arch Clin Exp Ophthalmol. 2002;240:983–988.
[CrossRef] [PubMed]SchuttF, BergmannM, HolzFG, KopitzJ. Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2003;44:3663–3668.
[CrossRef] [PubMed]BergmannM, SchüttF, HolzFG, KopitzJ. Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration. FASEB J. 2004;18:562–564.
[PubMed]RozanowskaM, Jarvis-EvansJ, KorytowskiW, BoultonM, BurkeJM, SarnaT. Blue light induced reactivity of retinal age pigment: in vitro generation of oxygen reactive species. J Biol Chem. 1995;270:18825–18830.
[CrossRef] [PubMed]BeattyS, KohH, PhilM, HensonD, BoultonM. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol. 2000;45:115–134.
[CrossRef] [PubMed]AndersonRE, KretzerFL, RappLM. Free radicals and ocular disease. Adv Exp Med Biol. 1994;366:73–86.
[PubMed]DaviesS, ElliottMH, FloorE, et al. Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells. Free Radic Biol Med. 2001;5:256–265.
WatzkeRC, SoldevillaJD, TruneDR. Morphometric analysis of human retinal pigment epithelium: correlation with age and location. Curr Eye Res. 1993;12:133–142.
[CrossRef] [PubMed]GaoH, HollyfieldJG. Aging of the human retina: differential loss of neurons and retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1992;33:1–17.
[PubMed]Panda-JonasS, JonasJB, Jakobczyk-ZmijaM. Retinal pigment epithelial cell count, distribution, and correlations in normal human eyes. Am J Ophthalmol. 1996;121:181–189.
[CrossRef] [PubMed]DenkW, StricklerJH, WebbWW. Two-photon laser scanning fluorescence microscopy. Science. 1990;248:73–76.
[CrossRef] [PubMed]ZipfelWR, WilliamsRM, WebbWW. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotech. 2003;21:1369–1377.
[CrossRef] MastersBR. Selected Papers on Multiphoton Excitation Microscopy. 2003;SPIE Optical Engineering Press Milestone Series MS 175. Bellingham, WA.
YehAT, NassifN, ZoumiA, TrombergBJ. Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence. Opt Lett. 2002;27:2082–2084.
[CrossRef] [PubMed]HanM, GieseG, ZicklerL, SunH, BilleJF. Mini-invasive corneal surgery and imaging with femtosecond lasers. Opt Express. 2004;12:4275–4281.
[CrossRef] [PubMed]HanM, GieseG, BilleJF. Second harmonic generation imaging of collagen fibrils in cornea and sclera. Opt Express. 2005;13:5791–5797.
[CrossRef] [PubMed]KönigK, BeckerTW, FischerP, RiemannI, HalbhuberKJ. Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes. Opt Let. 1999;24:113–115.
[CrossRef] HoptA, NeherE. Highly nonlinear photodamage in two-photo fluorescence microscopy. Biophys J. 2001;80:2029–2036.
[CrossRef] [PubMed]WatzkeRC, SoldevillaJD, TruneDR. Morphometric analysis of human retinal pigment epithelium: correlation with age and location. Curr Eye Res. 1993;12:133.
[CrossRef] [PubMed]HolzFG, BellmannC, StaudtS, SchüttF, VölckerHE. Fundus autofluorescence and development of geographic atrophy in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2001;42:1051–1056.
[PubMed]Schmitz-ValckenbergS, BültmannS, DreyhauptJ, BindewaldA, HolzFG, RohrschneiderK. Fundus autofluorescence and fundus perimetry in the junctional zone of geographic atrophy in patients with age-related macular degeneration. Invest Ophthalmol Vis Sci. 2004;45:4470–4476.
[CrossRef] [PubMed]WingGL, BlanchardGC, WeiterJJ. The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1978;17:601–607.
[PubMed]WeiterJJ, DeloriFC, WingGL, FitchKA. Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes. Invest Ophthalmol Vis Sci. 1986;27:145–152.
[PubMed]MastersBR, SoPT, BuehlerC, et al. Mitigating thermal mechanical damage potential during two-photo dermal imaging. J Biomed Opt. 2004;9:1265–1270.
[CrossRef] [PubMed]