Resonance Raman imaging of macular pigment distributions in the human retina

Abstract

We describe resonance Raman imaging (RRI) of macular pigment (MP) distributions in the living human eye. MP consists of the antioxidant carotenoid compounds lutein and zeaxanthin, is typically present in high concentrations in the healthy human macula relative to the peripheral retina, and is thought to protect this important central region from age-related macular degeneration. We demonstrate that RRI is capable of quantifying and imaging the spatially strongly varying MP distribution in the human retina. Using laser excitation of the MP molecules at 488nm, and sequential camera detection of light emitted back from the retina at the MP's strongest Raman peak position and at an off-peak position, RRI maps of MP are obtained at a resolution below 50microm within a fraction of a second per exposure. RRI imaging can be carried out with undilated pupils and provides a highly molecule-specific diagnostic imaging approach for MP distributions in human subjects.

Figures

Fig. 1

(Color online) Schematics of RRI system used to measure MP distributions in the living human eye and in excised retinal sample preparations. A 488 nm excitation beam from a solid-state laser is routed onto the retina via an optical fiber, collimating lens, L1, laser line filter, F1, achromatic beam expander, L2, dichroic beam splitter, BS1, and aperture, AP. Beam spot size on the retina is 3.5 mm in diameter. Light emitted from the retina is imaged onto the pixel array of a CCD camera with a 50 mm achromat. For RRI imaging, the light returned from the retina is first filtered with a combination of a narrowband tunable filter, F4, a bandpass filter, F5, and a notch filter, F3, effectively limiting the transmitted wavelength range to 528 nm, the spectral position of the resonance Raman response of MP under 488 nm excitation. In order to determine a correction for the background fluorescence, the light returned from the retina is filtered, in a second image, with a bandpass filter centered in the vicinity of the Raman peak at ~540 nm. Inset (a) shows the modification of the setup for measurements of excised eyecups, which are Raman imaged with the help of an angle-tunable filter, F4, tuned to two “on” and “off” Raman peak spectral positions near 528 nm. Inset (b) shows the modification for RRI imaging of retinal tissue sections through a microscope. Samples can be viewed with white-light illumination and translated with micrometer-scale precision.

Fig. 2

(Color online) Schematics of retinal layer system with indication of excitation and emission light intensities encountered in RRI of MP: OM, anterior optical media; MP, macular-pigment-containing layer; OS, photoreceptor outer segment layer; LP, lipofuscin-containing layer; IOM, light intensity originating from optical media fluorescence; IR, Raman response from MP; ILP, light intensity due to lipofuscin fluorescence (see text).

Fig. 3

(Color online) En face (a) and three-dimensional (b) pseudocolor-scaled RRI images of a dried drop of lutein spotted onto a PVDF substrate. The lutein solution had a physiological carotenoid concentration OD ≈0.8. The intensity of each pixel is color coded according to the scale shown in (a) and is displayed as a function of pixel position in the camera CCD array (1 pixel= 20 μm).

Fig. 4

(Color online) (a), (c) Two-dimensional, pseudocolor Raman images of 2 of the 11 donor eyecups imaged to establish a correlation between Raman- and HPLC-derived carotenoid levels; (b), (d) corresponding three-dimensional images. The color scale bar indicates the color coding of light intensities. The graph in (e) shows the correlation between optical intensities integrated over the macular regions of the eyecups and subsequently derived HPLC levels obtained for 8 mm diameter tissue punches centered on the macula (correlation coefficient R=0.92; p < 0.0001).

Fig. 5

(Color online) RRI results for MP distribution in living human eye. (a) Typical gray-scale image obtained after subtraction of fluorescence background from pixel intensity map containing Raman response and superimposed fluorescence background. (b) Pseudocolor-scaled, three-dimensional representation of gray-scale image. (c) Line plots along nasal–temporal (solid curve) and inferior–superior meridians (dashed curve), both running through the center of the MP distribution.

Fig. 6

(Color online) (a) Pseudocolor-scaled, three-dimensional MP RRI images obtained from four healthy subjects, demonstrating significant intersubject variations in MP levels, symmetries, and spatial extent. Note ringlike MP distribution with small central MP peak and overall low levels in one of the cases (lower left). All images are color coded with the same intensity bar. (b) Intensity plot profiles derived for each MP distribution from pixel intensity maps (22×1400 μm rectangles) running along nasal–temporal meridians through the center of the macula.

Fig. 7

(Color online) Images of MP distributions obtained for the same subject with (a) RRI and (b) fluorescence-based imaging. (c) Comparison of integrated MP densities obtained for 17 subjects with Raman- and fluorescence-based imaging methods. Vertical scale shows integrated MP densities derived from RRI images by integrating intensities over the whole macular region; horizontal scale shows corresponding densities derived via fluorescence imaging. A high correlation coefficient of R=0.89 is obtained for both methods, using a best fit that is not forced through zero. If the fit is forced through zero (not shown), one obtains a correlation coefficient R=0.80.

Fig. 8

Optical density in fovea versus thickness of retina in foveal pit, measured for eight healthy subjects. Thickness data were obtained from optical coherence tomography scans (see text).

Fig. 9

Pseudocolor RRI images of three subjects with ringlike MP distributions. (a) (Multimedia online; josaa.osa.org) A 57-year-old healthy male with MP distribution consisting of a narrow central peak and a surrounding strong, nearly rotationally symmetric distribution. MP levels in the ring are slightly higher than in the center and feature a noticeable disruption/offset at the “2 o’clock” position. (b) A 70-year-old female diagnosed with a mild form of dry AMD, showing a weak, broken-up ring structure with central high MP density and crosslike spokes. (c), (d) MP distributions in left (c) and right eye (d) of a 62-year-old female measured after detachment of the vitreous in the right eye. Six months prior to detachment, RRI images revealed the same ringlike MP pattern with a central spike in both eyes. Detachment of the vitreous apparently caused the formation of a double-peak MP structure inside the MP ring in this subject.