Imaging macular carotenoids and their related proteins in the human retina with confocal resonance Raman and fluorescence microscopy

Abstract

Lutein and zeaxanthin are highly concentrated at the central region of the human retina, forming a distinct yellow spot known as the macula lutea. The delivery and retention of the macular pigment carotenoids in the macula lutea involves many proteins, but their exact roles remain incompletely understood. In our study, we examined the distribution of the twelve known macular carotenoid-related proteins within the human macula and the underlying retinal pigment epithelium (RPE) using both fluorescence and Raman modes on our confocal resonance Raman microscope. Additionally, we assessed protein and gene expression through Western blot analysis and a single-cell RNA sequencing database. Our findings revealed that GSTP1, BCO2, and Aster-B exhibited distribution patterns similar to the macular carotenoids, with higher expression levels within the macular region compared to the periphery, while SR-BI and ABCA1 did not exhibit specific distribution patterns within the macula or RPE. Interestingly, LIPC, SR-BI's partner, accumulated specifically in the sub-foveal RPE. All three of these carotenoid transport proteins were found to be highly expressed in the RPE. These results offer valuable insights into the roles these proteins play in the formation of the macula lutea.

Keywords: BCO2; Carotenoid; Fluorescence; GSTP1; Macular pigment; Raman.

Fig. 1.. Optimization of Raman and Fluorescence measurements using confocal resonance Raman Microscopy.

To optimize the parameters for carotenoid testing by Raman and fluorescence for IHC of protein, we measured the Raman and fluorescence spectra of standard zeaxanthin (100 μM in methanol, black solid line) and Rhodamine red (5 μg/mL in PBS, grey dotted line) with the excitation of 473 nm and 532 nm lasers. The determined cursor positions were shown in red lines.

Fig. 2.. The distribution and expression of GSTP1 in the human macula and its underlying RPE.

(A) The distribution of GSTP1 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of GSTP1 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of GSTP1 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 3.. The distribution and expression of StARD3 in the human macula and its underlying RPE.

(A) The distribution of StARD3 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of StARD3 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of StARD3 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 4.. The distribution and expression of BCO1 in the human macula and its underlying RPE.

(A) The distribution of BCO1 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of BCO1 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of BCO1 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 5.. The distribution and expression of BCO2 in the human macula and its underlying RPE.

(A) The distribution of BCO2 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of BCO2 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of BCO2 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 6.. The distribution and expression of RPE65 in the human macula and its underlying RPE.

(A) The distribution of RPE65 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of RPE65 protein in in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of RPE65 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 7.. The distribution and expression of ABCA1 in the human macula and its underlying RPE.

(A) The distribution of ABCA1 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of ABCA1 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of ABCA1 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 8.. The distribution and expression of SR-B1 in the human macula and its underlying RPE.

(A) The distribution of SR-B1 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of SR-B1 protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of SR-B1 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 9.. The distribution and expression of LIPC in the human macula and its underlying RPE.

(A) The distribution of LIPC (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of LIPC protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of LIPC in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 10.. The distribution and expression of CD36 in the human macula and its underlying RPE.

(A) The distribution of CD36 (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of CD36 in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of CD36 in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 11.. The distribution and expression of LDLR in the human macula and its underlying RPE.

(A) The distribution of LDLR (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of LDLR protein in in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of LDLR in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 12.. The distribution and expression of Aster-B in the human macula and its underlying RPE.

(A) The distribution of Aster-B (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of Aster-B protein in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of Aster-B in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.

Fig. 13.. The distribution and expression of TTC39B in the human macula and its underlying RPE.

(A) The distribution of TTC39B (green) and carotenoids (red) in an 8 mm human macula and sub-macular RPE. Scale bar in white, represents 1 mm. (B) The expression of TTC 39B protein in in 8 mm punches of the human macula, peripheral retina, sub-macular RPE, and peripheral RPE detected by western blots. The gene expression of TTC39B in retinal (C) and RPE (D) cells from human macular (red) and peripheral (green) region. Plots were generated using the Iowa single-cell RNA sequencing database.