To demonstrate the performance of the system in terms of axial eye motion correction we recorded eight adjacent 3D data sets of the entire retinal depth with increasing eccentricities from the fovea (step size 0.5°). The eight data sets have been stitched together to cover an area of ~5°×1°. shows a “fly through” movie from the anterior part of the retina to the posterior part and the corresponding B-scan “fly through” movie of the same data set. Note that the dynamic focus ensures that the entire depth of the retina is in focus. As the first movie starts, individual nerve fiber bundles within the nerve fiber layer can be observed. As the coherence plane is moved further into tissue, small capillaries in the ganglion cell layer as well as at the top and bottom of the inner nuclear layer can be seen. Deeper into the tissue the external limiting membrane (faint signal) is followed by the junction between inner and outer segments of photoreceptors and the end tips of photoreceptors. Both layers show the cone mosaic. The last layer visible in the movie is the retinal pigment epithelium. The B-scan movie shows the excellent performance of the system to correct for axial eye motion correction.
Fig. 2 (Media 1) Frame number 65 of a movie retrieved from a 3D data set recorded with the instrument. The movie starts from the anterior part of the retina to the posterior part. Field of view: ~5°×1°. Imaging depth: ~393μm (in (more ...)
Fig. 3 (Media 2) Frame number 50 of a movie retrieved from the same data set as in . The movie starts from the inferior part of the retina to the superior part. Image size: 5° × 393μm (in tissue) Images are represented in a logarithmic (more ...)
Note that due to the large field of view shown in the movie (with limited memory size) the resolution of the original data was reduced and without zooming into the original data set individual cone photoreceptors are rather hardly visible. A slight mismatch in axial direction can be observed between individual 3D data sets. This mismatch varies in y-direction and is caused by a slightly changing entrance pupil (vertical position) of the imaging beam from data set to data set. This mismatch might be compensated using an A-scan based correction algorithm on the final data set.
In a next step we use the instrument to investigate an interesting observation (i.e. bright reflections from within the outer segments of photoreceptors that are visible in 3D OCT data sets) in more detail. Five healthy volunteers with good eye optics participated in this study. ) shows a full depth B-scan (1° scanning angle) reconstructed from a 3D data set to introduce the labeling of the different retinal layers used in this manuscript. As shown in a similar cone mosaic can be observed at the inner/outer segments junction of cone photoreceptors (IS/OS) and at the end tips of photoreceptors (ETPR). However, isolated bright reflections (BRs) can be observed between these layers i.e. within the outer segments of cone photoreceptors (c.f. . This observation has been made earlier also by other groups in OCT B-scans [23
]. Note that at some of these locations no backscattering intensity can be observed within the ETPR layer (c.f. circles in ). To test the reproducibility of the system we recorded several volumes at the same location within 5 minutes. shows SLO, en-face z-projection OCT as well as representative B-scans of exactly the same location and demonstrates the good reproducibility of our technique.
Fig. 4 Images of the human cone mosaic at ~4deg eccentricity. a) Overview B-scan image. ELM external limiting membrane, IS/OS junction between inner and outer segments of photoreceptors, OS outer segments photoreceptors, ETPR end tips photoreceptors, RPE retinal (more ...)
Fig. 5 Measurement series of the same location on the retina recorded within 5 minutes. Upper row: SLO images, middle row: representative B-scan images (location marked with a white line in the lower rows). The bright layers within the B-scan images are (from (more ...)
A good method to count the bright reflections (BR) would be to average all en-face images within the length of the OS (excluding IS/OS and ETPR layers) and to apply an intensity threshold on the resulting image. However, we found that this method does not take into account a varying cone outer segment length which can be observed at larger eccentricities from the fovea. Therefore we chose to count these BRs by an expert viewer who looked through all the reconstructed OCT B-scans for that purpose. With this procedure we measured the density of these reflection sites at different eccentricities from the fovea. Additionally we performed at each eccentricity a 2D Fourier transformation of all summated OCT en-face slices that show the cone mosaic to obtain Yellot's rings (YR) [25
]. shows a movie of the calculated FFT's for volunteer 2. The movie starts at the foveal center and ends at an eccentricity of 7° with a spacing of 1° between individual frames. While in the first two frames no ring can be observed a decrease of the radius of the ring can be observed in the rest of the movie. The maximum of the ring corresponds to the spatial frequency of the cone rows or modal frequency of cone mosaic [26
]. This frequency has to be multiplied by a factor (1/cos 30°) to convert the modal frequency into closest neighbor spacing. Assuming a hexagonal arrangement of the cones and a conversion factor of 291 μm for 1 degree scanning angle we calculated the cone density for each eccentricity. shows the result of both measurements. As indicated in ) the cone mosaic could be resolved down to 2° eccentricity from the fovea even without the use of adaptive optics for four volunteers. Closer to the fovea the packing density of the cones is very high and therefore YRs could not be observed. The measured decrease of cone density is in excellent agreement with data known from histology [28
] or AO-OCT [29
]. The border of 2° eccentricity between resolvable cones and non resolvable cones is in excellent agreement with the theoretical resolution of the system (~5μm) and the expected row to row spacing of 5.5μm that is obtained from histology data at this eccentricity. To visualize the performance of the system shows the cone mosaic at 2 degrees eccentricity in a comparison between SLO and OCT (integrated over the entire imaging depth). Note that on the right hand side of the images (closer to the fovea) the cones become hardly separated especially in the OCT image indicating the resolution limit of the system.
(Media 3) Frame No.3 (corresponding to 2° eccentricity) of a movie showing the calculated FFT's of the cone mosaic measured in volunteer 2.
Fig. 7 a) Measured cone density of five volunteers at different eccentricities from the fovea (black line: data from Ref . b) Measured bright reflections (BR) within the cone outer segments at different eccentricities (Each color corresponds to a different (more ...)
The cone mosaic imaged at 2 degrees eccentricity from the fovea. a) depth integrated OCT image, b) SLO image (field of view: ~1°×1°).
However, the measured density of the BRs follows a different dependence on the eccentricity from the fovea (c.f. ). In four volunteers we observed an increase of BR with eccentricity with a maximum of BR density at ~4° eccentricity. At larger eccentricities this density decreases again. One volunteer showed almost constant BR density with eccentricity. We want to emphasize here that even though we are not able to resolve individual cones at eccentricities smaller than 2° it is still possible to count the BR's at these eccentricities provided that these are quite isolated (which is the case at larger eccentricities).
Although we counted every BR for the measurement above we observed differing arrangements of these spots with respect to the normal reflections from the IS/OS junction and ETPR layer. illustrates five different possibillities: a) Three distinct spots can be observed but the spot from within the outer segment has the highest intensity, b) Three distinct spots with equal intensity, c) only one highly reflecting spot, d) one spot within the outer segments, one within the ETPR layer, e) one spot within the IS/OS junction and one within the outer segments (no reflection from the ETPR).
Different arrangements of BR (marked with an ellipse) observed at 4° eccentricity (see text for explanation).