In vivo measurement of organelle motility in human retinal pigment epithelial cells

Abstract

Retinal pigment epithelial (RPE) cells are well known to play a central role in the progression of numerous retinal diseases. Changes in the structure and function of these cells thus may serve as sensitive biomarkers of disease onset. While in vivo studies have focused on structural changes, functional ones may better capture cell health owing to their more direct connection to cell physiology. In this study, we developed a method based on adaptive optics optical coherence tomography (AO-OCT) and speckle field dynamics for characterizing organelle motility in individual RPE cells. We quantified the dynamics in terms of an exponential decay time constant, the time required for the speckle field to decorrelate. Using seven normal subjects, we found the RPE speckle field to decorrelate in about 5 s. This result has two fundamental implications for future clinical use. First, it establishes a path for generating a normative baseline to which motility of diseased RPE cells can be compared. Second, it predicts an AO-OCT image acquisition time that is 36 times faster than used in our earlier report for individuating RPE cells, thus a major improvement in clinical efficacy.

Conflict of interest statement

The authors declare that there are no conflicts of interest related to this article. Disclaimer: The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the US Department of Health and Human Services.

Figures

Fig. 1

Assessing motility dynamics in RPE and cone cells in a 26-year-old subject (S2) at 7° temporal retina using AO-OCT imaging. (A) Schematic depicts the three layers analyzed: (1) ONL, (2) cone (IS/OS + COST), and (3) RPE. En face images were extracted from the same registered and averaged AO-OCT volume at depths of (B) ONL, (C) cone (projection of cone IS/OS and COST), and (D) RPE. Average was over 45 registered AO-OCT volumes. Each RPE cell in (D) is represented by a Voronoi cell in (E). (F) Shown is a magnified view of Voronoi cells in the blue box superimposed on the RPE map in (E). A motility function (CF) was calculated for each Voronoi cell—an example for one RPE cell is diagrammed at bottom of (F)—and then averaged across cells to increase SNR.

Fig. 2

Averaged and registered RPE images for the seven subjects imaged at 7° temporal to the fovea. N is the total number of images averaged. TI denotes mean time intervals between RPE images, which were selected one per AO-OCT video. 2-D power spectra are superimposed at bottom right of each en face image.

Fig. 3

Motility dynamics measured at three retinal layers in seven subjects. (A) Representative raw motility function from S2 (Experiment 3) before normalization to the cone layer. Error bars represent standard deviation across 475 RPE cells. Colors represent measurements taken at three retinal layers: ONL (blue), Cone (IS/OS + COST) (red), and RPE (green). Colors in (A) also apply in (B). (B) Motility function is normalized to cone layer to remove residual eye motion and system errors. (C) Normalized RPE motility measurements are shown for the seven subjects. Scatterplots with ♦ symbols were from Experiment 1 with short video durations of 1.8 s, and scatterplots with ● symbols were from Experiments 2 and 3 with long video durations of ~9 s (see Table 2 for acquisition parameters). The gap between ~10 s and ~100 s for this data set is due to the time interval between two consecutive videos. (see Section 2.2). Fitted time constants are given in the key for each subject in each experiment.

Fig. 4

Clarity of the RPE cell mosaic depends on the time interval between acquired AO-OCT images. En face images are averages of 35 volumes with an average time interval of (A) 0.23 s, (B) 7.8 s, and (C) 64.6 s in subject S2. The corresponding histograms of time interval (TI), 2-D power spectra and magnified sub-images (50 μm × 50 μm) indicated by the blue box in (A-C) are shown below each image. Note: Images of (A) and (B) are from the same patch of the retina; (C) is from a slightly different patch.

Fig. 5

(A) Signal-to-noise ratio (SNR) of RPE mosaic fundamental frequency as a function of AO-OCT images averaged from subject S2. The RPE mosaic fundamental frequency and noise were determined from the power spectrum of the registered, averaged images. Images were acquired at averaged TI of 0.23 s (blue), 7.8 s (black) and 64.6 s (red). RPE en face images of: (B) single volume (N = 1), (C) average of 35 volumes with averaged TI = 0.23 s, (D) average of 35 volumes with TI = 7.8 s, and average of 5 volumes with averaged (E) TI = 0.18 s, and (G) TI = 6.16 s. The number of effective volumes that generate each image pixel in (E) and (G) varies between 0 and 5 depending on eye motion as shown in (F) and (H), respectively. The fast B-scan direction of the AO-OCT system is horizontal.

Fig. 6

Motion-evoked speckle changes with depth in the RPE-BM complex. (A) Averaged AO-OCT B-scan of S2 shows distinct hyper- and hypo-reflective bands in the outer retina. Four depths are marked on the superimposed averaged A-line profile that represent apical, middle, and basal sub-layers of the RPE and the BM layer. The color-coded CFs at the four corresponding depths are shown in (B). The average time constants of four subjects (S2, S5-S7) are shown in (C). Error bars denote standard deviation. Also shown are the corresponding en face images of the averaged volume (D-G) and single frame from the reference volume (H-K) of S2.

Fig. 7

Representative eye motion measured in two series of 35 AO-OCT images in three dimensions (3-D) from S2 with different time intervals (magenta: TI = 0.23 s, and blue: TI = 7.8 s). To better visualize the individual components, the 3-D motion is projected into the x-y, x-z, and y-z planes. Green point denotes the reference image coordinate: (x, y, z) = (0, 0, 0).