2.1.

Scanning Protocol

We modulated both raster and circular scans, which are commonly used in vis-OCT,¹⁶ to test our speckle reduction method. Figure 1 illustrates the modulated raster scan [Figs. 1(a) and 1(b)] and modulated circular scan [Figs. 1(c) and 1(d)]. As shown in Figs. 1(a) and 1(c), we define the B-scan axis as the direction along which a traditional cross-sectional image would be acquired without modulation. We define the orthogonal axis as the direction orthogonal to the B-scan axis on the two-dimensional scanning plane. Movement along the orthogonal axis [arrow 1 in Figs. 1(b) and 1(d)] occurs in $n$ equidistant steps, where $n$ is the number of speckle-uncorrelated A-lines to be locally averaged. Each translation of the galvanometer [red dots in Figs. 1(b) and 1(d)] is discrete, synchronized with the spectrometer camera exposure, and implemented entirely via software control without additional hardware or moving parts. This avoids a complex synchronization procedure or risk of desynchronization between the beam path and the camera exposure when using an external scanner.¹⁰ The centroids of each spot generating an A-line are separated by a distance $d$ along the orthogonal axis [Fig. 1(b)]. After $n$ translations in this direction, the beam is shifted along the B-scan axis [arrow 2 in Figs. 1(b) and 1(d)], followed by a reversed scan along the orthogonal axis [arrow 3 in Figs. 1(b) and 1(d)]. Such modulation superimposes a rectangular wave on the B-scan axis, where each rising and falling edge of each rectangle contains $n$ speckle-uncorrelated A-lines. While other modulation shapes such as sinusoidal or triangular are possible, we chose rectangular to best preserve lateral resolution along the B-scan axis. During acquisition, several parameters, including $n$, $d$, and imaging FOV are adjustable. We investigate how to obtain an optimal $d$ in Sec. 3.1.

Fig. 1

Illustrations of speckle-reduction scanning protocols. (a) Overall illustration of the relationship between the B-scan axis and the orthogonal axis in the modulated raster scan; (b) detailed illustration of the A-line acquisition sequence in the modulated raster scan. Here, $d$ is the distance between two adjacent A-lines along the orthogonal axis. The arrows 1, 2, and 3 highlight the trajectory of galvanometer motion. (c) Overall illustration of the relationship between the B-scan axis and the orthogonal axis in the modulated circular scan; (d) detailed illustration of the A-line acquisition sequence in the modulated circular scan.

We averaged all $n$ A-lines in the orthogonal direction along each rectangular edge [Figs. 1(b) and 1(d)] to generate a single speckle-reduced A-line (srA-line). For a desired sampling density of $m$ srA-lines per speckle-reduced B-scan (srB-scan), the total number of camera acquisitions per srB-scan is $n\times m$. Each consecutive srA-line in an srB-scan can then be calculated as

Since an srB-scan increases imaging time over normal B-scan acquisition by a factor of $n$, it is important to collectively limit $n$, $m$, and the number of total srB-scans to prevent overly long imaging time. First, all sampling numbers were selected in powers of 2 to support fast graphics processing unit data processing. Next, we limited all imaging experiments to 8192 total A-lines per srB-scan. Given a camera exposure time of $40\mu \mathrm{s}$, which is required for sufficiently high SNR, an srB-scan could be acquired in 328 ms, an upper limit for reducing bulk motion artifacts (satisfying Nyquist criterion of 500 ms for eye microsaccades of $\sim 1\mathrm{Hz}$).²⁰ Furthermore, we chose to limit the total image acquisition time to $\sim 5\mathrm{s}$ to prevent patient fatigue and discomfort. This limited the total amount of srB-scans per acquisition to 16 (5.25 s total acquisition time). In our experimental human imaging system (Sec. 2.3.2), we maximized lateral sampling density in a raster scan without spot overlap, where $m=1024$ srA-lines and $n=8$ averages. The parameters $n$, $m$, and $d$ can be easily modified for different experimental conditions.

The srB-scan averages along the same locations as n spatially separated B-scans, each of m A-lines. However, in direct B-scan averaging, each A-line at a particular lateral position is delayed temporally by the scanner’s fly-back time. This results in a total sampling period of $n\times m\times t$, where $t$ is the camera exposure time for each A-line. In our method, modulation removes the wait for scanner fly-back, thereby reducing the total sampling period to $n\times t$ for each srA-line. Using our experimental parameters ($m=1024$, $n=8$, $t=40\mu \mathrm{s}$), we reduced the sampling period from 328 ms to $320\mu \mathrm{s}$ for each srA-line and increased the srA-line rate from 3 to 3125 Hz. Constant, involuntary retinal motions²⁰ can occur at frequencies up to 90 Hz with amplitudes up to 40 arc sec (equivalent to $0.011\text{-}\mu \mathrm{m}$ change in sampling location per $40\text{-}\mu \mathrm{s}$ camera exposure in the human retina). This leaves the possibility of only $0.088\mu \mathrm{m}$ of movement during an srA-line, which is insignificant when compared with the micron-order lateral and axial resolutions in OCT. Therefore, the improved srA-line rate is highly significant.

2.2.

Metrics to Evaluate Image Quality Improvement

We used contrast-to-noise ratio (CNR) and equivalent number of looks (ENL)²¹ to evaluate image quality improvement after speckle reduction. CNR measures how well the sample feature can be discerned from the surrounding background. Mean intensity and variance from both the image background and the sample feature are included to account for two separate noise components: intrinsic OCT background noise and speckle. Since the optical properties of different features vary, we calculated the CNR (dB) in confined region of interests (ROIs) as

ENL is the squared inverse of the speckle contrast and measures the smoothness and homogeneity within an ROI. We calculated ENL as

We compared CNR and ENL in srB-scans with a “reference” B-scan from the same location as an srB-scan. A reference B-scan included 8192 A-lines acquired along the B-scan axis with a $40\text{-}\mu \mathrm{s}$ camera exposure. Every eight consecutive A-lines were averaged, resulting in a final sampling density of 1024 averaged A-lines per reference B-scan. This operation was equivalent to acquiring an srB-scan without modulating the scanner along the orthogonal axis. Because of high sampling density along the B-scan axis, averaged speckle patterns were still highly correlated, preventing reduction of speckle. However, background noise was equally suppressed in reference B-scans and srB-scans because they used the same amount of temporal averaging. We compared the CNR and ENL values between the reference B-scan and srB-scans to evaluate the effectiveness of speckle reduction.

2.3.

Data Acquisition

We tested our speckle reduction protocol in both mouse and human retinas using two prototype systems developed at Northwestern University. In addition, we further tested our speckle reduction method in humans in a clinical setting using a commercial vis-OCT system (Aurora X1, Opticent Health), where optical engineering expertise was unavailable. We directly implemented the modulated scanning protocol in that system without additional calibration, alignment, or changes to the photographer’s workflow.

2.4.

Initial Calibration for Orthogonal Spot Separation

A calibration procedure was needed for coarse determination of optimal spot separation, d, along the orthogonal axis. Since CNR is associated with the ability to discern features from noise, we used it as the primary indicator for image quality. In theory, an increased $d$ increases the decorrelation of the speckle patterns between adjacent orthogonal A-lines. After averaging, speckle is maximally reduced when the averaged patterns are entirely uncorrelated.¹⁰ However, if $d$ is too large, we will lose structural similarity between orthogonal A-lines, which can result in image blurring. We investigated the impact of modulation distance on CNR by imaging a model mouse eye using both raster and circular scans. The model eye was made from a silica bead (diameter: 3.15 mm). We attached two layers of tape and paper with an ink pattern to the bottom of the bead to simulate the retinal layers. Using the rodent vis-OCT system, we reached a $1/e^2$ spot size of $\sim 5.5\mu \mathrm{m}$ on the tape layers through the bead. We then varied the $d$ value from 0 to $13.75\mu \mathrm{m}$ in 16 steps and acquired an srB-scan after each step. We calculated CNR from three ROIs in the top tape layer and averaged them to determine the impact of the $d$ value on image quality.

3.1.

Impact of Modulation Distance on Image Quality

The results to identify an optimal $d$ value are shown in Fig. 2. Figures 2(a) and 2(b), respectively, show how CNR values vary as a function of $d$ in imaging the model eye using modulated raster and circular scans. When $d$ is increased from 0 to $13.75\mu \mathrm{m}$ in both scans, CNR reaches its maximum at $d=6\mu \mathrm{m}$. Figures 2(c) and 2(e) show raster srB-scans with $d=0$ and $6\mu \mathrm{m}$, respectively. Figures 2(d) and 2(f) show magnified views of the two highlighted images [yellow boxes in Figs. 2(c) and 2(e)], respectively. The srB-scan with $d=6\mu \mathrm{m}$ [Fig. 2(f)] shows a smoother intensity distribution within each layer and much improved discrimination between the tape and the paper layers, as compared with the unmodulated scan [Fig. 2(d)]. Figures 2(g)–2(j) show the similar comparison in the circular scan, where the speckle-reduced circular scan with $d=6\mu \mathrm{m}$ demonstrates improvement in image quality.

Fig. 2

Speckle-reduction test in the model mouse eye. (a) Change of averaged CNR as a function of d in the modulated raster scan; (b) change of averaged CNR as a function of $d$ in the modulated circular scan. (c) An srB-scan image of the model mouse eye acquired using modulated raster scan with $d=0\mu \mathrm{m}$. The structures corresponding to the two tape and one paper layers are highlighted by the arrows. (d) Magnified view of the region highlighted by the box in panel (c). (e) An srB-scan image of the model mouse eye acquired using modulated raster scan with $d=6\mu \mathrm{m}$. (f) Magnified view of the region highlighted by the box in panel (e). (g) An srB-scan image of the model mouse eye acquired using modulated circular scan with $d=0\mu \mathrm{m}$. (h) Magnified view of the region highlighted by the box in panel (g). (i) An srB-scan image of the model mouse eye acquired using modulated circular scan with $d=6\mu \mathrm{m}$. (j) Magnified view of the region highlighted by the box in panel (i). All images are plotted with identical color bar. (k) Fitted pixel-intensity histograms within the tape layer 1 acquired by modulated raster scans with $d=0$ and $6\mu \mathrm{m}$; (l) fitted pixel-intensity histograms within the tape layer 1 acquired by modulated circular scans with $d=0$ and $6\mu \mathrm{m}$.

Figures 2(k) and 2(l), respectively, show the pixel intensity histograms from the tape layer 1 [highlighted in Fig. 2(c)] in the raster and circular scans. In both scan patterns, the intensity histograms changed from a broad, right-skewed distribution when $d=0\mu \mathrm{m}$ to a lower-variance, nearly centrosymmetric distribution when $d=6\mu \mathrm{m}$. These results agree with the expected change in pixel intensity distribution from Rayleigh distribution to Poisson distribution after speckle reduction.¹⁴

As shown in Figs. 2(a) and 2(b), we used a $-0.25\text{-}\mathrm{dB}$ drop in CNR to determine the range of acceptable d values, which gives $d_{\text{min}}=5.1\mu \mathrm{m}$ and $d_{\text{max}}=7.8\mu \mathrm{m}$. This range is helpful for human retinal imaging, where the eye shape, optical properties, and scanning location may differ among subjects. We also noted that the optimal $d=6\mu \mathrm{m}$ is approximately equal to the estimated spot size of $\sim 5.5\mu \mathrm{m}$ on the retina. This suggests that adjacent spots along the orthogonal axis should be as close as possible without spatial overlap. This result is consistent with the notion that spatial overlapping provides correlated speckle patterns. This result also suggests that it is acceptable to estimate the optimal $d$ using the OCT focal spot size on the sample. These considerations are not expected to change in the living human eye, where local movement during a single srA-line ($0.088\mu \mathrm{m}$) is significantly less than $d$.

We adjusted the $d$ value within the identified range in rodent and human imaging to accommodate different eye conditions. Since we control the $d$ value by the galvanometer angle, we identified optimal angular step size along the orthogonal axis in different experimental conditions. For mouse imaging, the optimal angular step sizes were 0.175 deg and 0.15 deg, which correspond to $d$ values of 6.3 and $5.4\mu \mathrm{m}$, respectively, in raster and circular scans. For human imaging, the optimal angular step sizes were 0.025 deg and 0.02 deg, which correspond to $d$ values of 7 and $5.9\mu \mathrm{m}$, respectively, in raster and circular scans.

3.2.

Speckle Reduction in the Mouse Retina

Figure 3 shows the speckle reduction results in a mouse retina using a raster scan. Figures 3(a) and 3(b) are the reference B-scan and srB-scan images, respectively. The imaged retina in the srB-scan has a smoother, less grainy appearance that provides a clearer differentiation between anatomical layers. We selected six ROIs from the inner plexiform layer [IPL, highlighted by (a1) and (b1)], outer nuclear layer [ONL, highlighted by (a2) and (b2)], and outer retinal layer [ORL, highlighted by (a3) and (b3)] to quantify quality improvement. Figures 3(a1)–3(a3) and Figs. 3(b1)–3(b3) show the magnified views of the six selected ROIs and Table 1 shows the quantitative comparisons of CNR and ENL values from these ROIs. Speckle reduction is particularly helpful in the ORL, where a small gap near RPE and BM layers is revealed [Fig. 3(b3)], which is not visible in the reference B-scan image [Fig. 3(a3)]. The capability to differentiate RPE and BM may add significant value to various preclinical studies using mouse models.

Fig. 3

Speckle-reduction test in mouse retina using modulated raster scan. (a) Reference raster B-scan image. Three ROIs from IPL, ONL, and ORL are highlighted by (a1), (a2), and (a3), respectively. The size of each ROI is $140\mu \mathrm{m}(\text{lateral})\times 20\mu \mathrm{m}(\text{axial})$. (b) The corresponding srB-scan image. The same three ROIs are highlighted by (b1), (b2), and (b3), respectively. (a1)–(a3) The magnified views of the three highlighted ROIs in panel (a). (b1)–(b3) The magnified views of the three highlighted ROIs in panel (b). CNR and ENL values are calculated from all the selected ROIs. All images are plotted with identical color bar.

Table 1

Image quality metric values from the ROIs in the mouse retina shown in Figs. 3 and 4.

Figure 4 shows the speckle reduction results in a mouse retina using circular scan. Figures 4(a) and 4(b) are the reference B-scan and srB-scan images, respectively. Again, the srB-scan image improved the overall image quality with better differentiated fine anatomical features. We also selected six ROIs from IPL [highlighted by (a1) and (b1)] and retinal blood vessels [highlighted by (a2), (b2), (a3), and (b3), respectively] for quantitative evaluation.

Fig. 4

Speckle-reduction test in mouse retina using modulated circular scan. (a) Reference circular B-scan image. Three ROIs from IPL and two vessels are highlighted by (a1), (a2), and (a3), respectively. The size of each ROI is $140\mu \mathrm{m}(\text{lateral})\times 20\mu \mathrm{m}(\text{axial})$. (b) The corresponding srB-scan image. The same three ROIs are highlighted by (b1), (b2), and (b3), respectively. CNR and ENL values are calculated from all the selected ROIs. All images are plotted with identical color bar.

Table 1 compares the CNR and ENL values from the selected ROIs in both raster and circular scans. In each scan mode, we see increased metric values from the ROIs in the srB-scan images. For raster scans, the ROIs in the IPL, ONL, and ORL show 2.35, 1.84, and 1.32 dB respective improvements in CNR, and 3.1, 2.53, and 1.84 times respective improvements in ENL. For circular scans, the ROIs in the IPL and two vessels show 1.84, 1.90, and 1.11 dB respective improvement in CNR, and 2.69, 2.56, and 1.63 times respective improvements in ENL. CNR and ENL improvements for the ORL in the raster scan and second vessel in the circular scan are slightly lower than other improvements. This is because some of the image background is unavoidably included in the ROI, artificially contributing low pixel intensities to ${\mu }_i$ in the metrics.

3.3.

Speckle Reduction in the Human Retina

We accomplished speckle reduction in the human retina using both the laboratorial prototype¹⁸ and a clinical vis-OCT system. Unlike mouse imaging, in which retinal motion can be minimized and images can be acquired over an extended period, human imaging usually suffers from severe retinal motions and image acquisition needs to complete within few seconds. For vis-OCT, retinal motion can be much stronger as described in Sec. 1.

Figure 5 shows the speckle reduction results using raster scan in a human retina (22-year-old male volunteer). Figures 5(a) and 5(b) are reference B-scan and srB-scan images superior to the optic disk, respectively. The srB-scan is smoother and less grainy in appearance than the reference B-scan, increasing visibility of the retinal layers. Improved image quality here is consistent with that in the mouse retina [Fig. 3(b)]. We selected six ROIs from the nerve fiber layer [NFL, highlighted by (a1) and (b1)], ganglion cell layer [GCL, highlighted by (a2) and (b2)], and ORLs [highlighted by (a3) and (b3)] to quantify quality improvement. Figures 5(a1)–5(a3) and 5(b1)–5(b3) show the magnified views of the six selected ROIs and Table 2 shows the quantitative comparisons of CNR and ENL values from these ROIs. Of particular note is the increased clarity of ORL in the srB-scan [Fig. 5(b3)]. Unlike the reference B-scan [Fig. 5(a3)], the shape and boundaries of the rod outer segment tips (ROST), RPE, and BM become clearly discernable from one another. The thickness of BM is measured as $\sim 3\mu \mathrm{m}$ and is resolved in the whole image without blur or distortion. The average measured thickness of BM in the human eye is $\sim 2$ to $5\mu \mathrm{m}$,²³ which is consistent with our measurement. The distinct separation between the BM and the RPE, as shown in Fig. 5(a3), may open up new window to investigate macular degeneration, where initial pathological alterations are hypothesized to start from BM.^23,24 Finally, we note a shadow caused by a small blood vessel as highlighted by the arrows in both the reference B-scan [Fig. 5(a3)] and the srB-scan [Fig. 5(b3)] images in ORL. It is measured as 2 pixels laterally or $\sim 14\mu \mathrm{m}$ in width. This feature is better resolved in the srB-scan image, indicating that lateral resolution has been well preserved after speckle reduction.

Fig. 5

Speckle-reduction test in human retina using modulated raster scan. (a) Reference raster B-scan image. Three ROIs from NFL, GCL, and ORL are highlighted by (a1), (a2), and (a3), respectively. The size of each ROI is $430\mu \mathrm{m}(\text{lateral})\times 23\mu \mathrm{m}(\text{axial})$. (b) The corresponding srB-scan image. The same three ROIs are highlighted by (b1), (b2), and (b3), respectively. (a1)–(a3) The magnified views of the three highlighted ROIs in panel (a). (b1)–(b3) The magnified views of the three highlighted ROIs in panel (b). CNR and ENL values are calculated from all the selected ROIs. The bottom three anatomical layers ROST, RPE, and BM are highlighted in panel (b3). The arrows in (a3) and (b3) highlight the same blood vessel shadow. All images are plotted with identical color bar.

Table 2

Image quality metric values from the ROIs in the human retina from Figs. 5 and 7.

Repetitive B-scan averaging is not trivial due to retinal motion, which often leads to image blurring even after registration. We overcame this challenge and showed that our speckle reduction method is robust against retinal motion in Fig. 6. We acquired eight repeated raster B-scans (each containing 1024 A-lines) from the same anatomical location and volunteer, as shown in Figs. 5(a) and 5(b). All the B-scans were axially and laterally registered using an fast Fourier transform based cross-correlation algorithm.²⁵ The averaged B-scan image [Fig. 6(a)] shows blurred anatomical layers in both the inner retina and the outer retina due to motion. Figure 6(b) shows a magnified view of the region highlighted by the box in Fig. 6(a). Two A-lines from the locations highlighted by lines 1 and 2 in Fig. 6(b) are shown in Fig. 6(c). A-line 1 reveals five anatomical layers in the outer retina, notably with reduced contrast near the RPE. A-line 2 fails to resolve any anatomical features. Since vis-OCT offers an axial resolution of near $1\mu \mathrm{m}$, small misalignments in B-scan averaging may lead to much severer blurring. The image quality shown in Fig. 6(a) is representative of most averaged B-scan images acquired by vis-OCT using similar scan parameters. Figure 6(d) shows a magnified view of the same anatomical position from an srB-scan image, where all anatomical layers are clearly resolved across the whole image. The same A-line locations from Fig. 6(b) are highlighted in Fig. 6(d) (by 3 and 4). Figure 6(e) shows A-line 3 and A-line 4, confirming that all ORLs are well resolved despite retinal motion.

Fig. 6

Directly comparing averaged B-scan with srB-scan images from human retina. (a) Image scan from the same location as shown in Fig. 5(b) after averaging eight B-scans. (b) Magnified view of the outer retina region as highlighted in panel (a). (c) Two A-lines from the positions highlighted by 1 and 2 in panel (b). (d) Magnified view of the same outer retina region from the srB-scan shown in Fig. 5(b). Five anatomical layers are labeled. (e) Two A-lines from the positions highlighted by 3 and 4 in panel (d). All plotted A-lines are averaged three times laterally to reduce variation. All images are plotted on the same contrast scale as used in Fig. 5.

We also demonstrate speckle reduction in circular scan in the human retina (Fig. 7). Figures 7(a) and 7(b) show a reference B-scan image and an srB-scan image, acquired at the same anatomical location, respectively. We selected six ROIs from the same locations as in Fig. 5, including the NFL [highlighted by (a1) and (b1)], GCL [highlighted by (a2) and (b2)], and ORL [highlighted by (a3) and (b3)] to quantify quality improvement. Figures 7(a1)–7(a3) and 7(b1)–7(b3) show magnified views of the six selected ROIs and Table 2 shows the quantitative comparisons of the CNR and ENL values from these ROIs. Similar to the ORL in the raster srB-scan [Fig. 6(b3)], the ORL in the circular srB-scan [Fig. 7(b3)] shows distinct separation between BM, RPE, and ROST. In the reference B-scan image [Fig. 7(a3)], however, boundaries of these anatomical layers are not easily differentiated due to speckles. To the best of our knowledge, this is the first demonstration of speckle-reduced imaging in a circular pattern using localized scan modulation in the human retina.

Fig. 7

Speckle-reduction test in human retina using modulated circular scan. (a) Reference circular B-scan image. Three ROIs from NFL, GCL, and ORL are highlighted by (a1), (a2), and (a3), respectively. The size of each ROI is $900\mu \mathrm{m}(\text{lateral})\times 40\mu \mathrm{m}(\text{axial})$. (b) The corresponding srB-scan image. The same three ROIs are highlighted by (b1), (b2), and (b3), respectively. (a1)–(a3) The magnified views of the three highlighted ROIs in panel (a). (b1)–(b3) The magnified views of the three highlighted ROIs in panel (b). CNR and ENL values are calculated from all the selected ROIs. The bottom four anatomical layers cone outer segment tips (COST), ROST, RPE, and BM are highlighted in panel (b3). All images are plotted with identical color bar.

Table 2 compares the CNR and ENL values from the selected ROIs in the human retina for both raster and circular scans. In each instance, we see increased metric values in the srB-scan ROIs. Raster scans show an improvement in CNR of 2.25, 2.00, and 1.86 dB, in the NFL, GCL, and ORL, respectively. Corresponding ENL improvements are 2.87, 2.72, and 2.52 times, respectively. Circular scans show an improvement in CNR of 2.08, 1.92, and 1.64 dB, in the NFL, GCL, and ORL, respectively. Here, corresponding ENL improvements are 2.77, 2.11, and 1.81 times, respectively. Again, we attribute the slightly lower metric value increases in the ORL to the unavoidable inclusion of image background in the ROI, artificially contributing low pixel intensities to ${\mu }_i$ in the metrics.

3.4.

Speckle Reduction Test in Clinical Environment

A clinical photographer without technical knowledge of the scanning protocol independently verified speckle reduction in the human retina using a commercial vis-OCT system. The photographer acquired images using the same procedure as acquiring normal raster scan images and received no additional training. Quality improvement in the clinical speckle-reduced images is comparable to the lab tests (Sec. 3.3). Figures 8(a) and 8(b), respectively, show a reference B-scan and an srB-scan of the macula from a 37-year-old female volunteer. In the reference B-scan image, speckle particularly distorts the ORL, as shown in Fig. 8(c), preventing the delineation of fine anatomical structures, such as the RPE and BM. As a comparison, all five ORLs, including the RPE and BM, are clearly resolved in the magnified srB-scan image, as shown in Fig. 8(d). Quantitatively, the improvements in CNR and ENL from similar ROIs are comparable with what we achieved in lab tests.

Fig. 8

Speckle-reduction test in human retina in clinical environment using modulated raster scan. (a) Reference raster B-scan image; (b) the corresponding srB-scan image; (c) magnified view of the region highlighted in panel (a); and (d) magnified view of the region highlighted in panel (b). The five anatomical layers, IS/OS, COST, ROST, RPE, and BM, are labeled. All images are plotted with identical color bar.