Schematics of trajectories of SS and MS photons with different focus depths; i.e., different defocus amounts. No matter the focal position, the SS photon is scattered only once and hence has the same path length. This results in the consistent phase of the SS signal after computational refocusing. Meanwhile, the trajectories of MS photons are scrambled by changes in the focal position. Hence, the phase of the MS signal is randomized even after the computational refocusing.

Schematic (a) and photograph (b) of a scattering phantom. The phantom comprises glass slips A and D, a scattering layer B, which is a mixture of polystyrene micro-particles and ultrasound gel, and a glass plate C embedded in the scattering layer. The glass plate C provides a scattering-free area. A postmortem zebrafish at 30 days post fertilization (dpf) was used as a biological sample. The sample is shown in a color photograph (c) and a wide-field-of-view OCT intensity projection (d). The orange box around the belly region denotes the measurement area in the validation study of the MFA method.

(a)–(c) and (d)–(f) show the B-scans and en face images of the phantom, respectively. In (a)–(c), the orange arrowheads denote the approximate focal positions, and the yellow boxes denote a deep region of the scattering layer. In (d)–(f), the magnification insets are the en face images of a small region in the scattering layer containing several scatterers. In (d), the red circles denote the scatterers selected to compute the signal intensity. The blue line indicates a region with 2 × 200 A-lines in the glass plate region used to compute the background intensity for the SBR analysis, and their locations in the B-scans are indicated by the blue bracket in (a). White arrows in (d)-(f) indicate a low-intensity scatterer signal, which is dimmed in (f). It could be the speckle caused by the MS signal. (g) shows the intensity depth profiles that are averaged by the A-lines at the en face locations denoted by the blue line in (d). The 0-mm depth refers to the top surface of the cover glass on the top, and the green arrowhead denotes the depth in air where the intensity is 0 dB. The orange arrowheads in (a) and (g) denote the depth where the en face images are taken.

Cross sectional and en face images of the postmortem zebrafish. In (a)–(c), the orange arrowheads denote the approximate focal positions, and the magnification insets are supposed to show notochord structure. The black arrows in (a)–(c) and (g)–(i) may indicate the myosepta. In (d)–(f), white dashed areas denote the belly region, and blue dashed ellipses mark fine structures observed in the muscle region. The green arrowheads in (d)–(f) and (j)–(l) may indicate the outer layer of the swim bladder. In (g)–(i), yellow dashed circles denote several hyper-scattering spots observed in the belly region. In (j)–(l), white ellipses denote unidentified structures that are better contrasted in the MFA image, and small red boxes indicate two selected regions for computing SBR. At all depths, the MFA method has the images with best contrast among the three methods.

(a) and (b) are B-scans at the same location of a phantom volume without and with refocusing, respectively. (c) and (d) show representative spatial auto-correlation functions of the linear-scaled images at a depth [dashed vertical lines in (e)] along both directions without and with applying refocusing, respectively, from which the FWHMs are measured. (e) shows the FWHMs along the depth, which are considered to be proportional to the speckle size. The first and second rows present the results without and with computational refocusing, respectively. Each column presents the results for a volume measured with different focal positions. The blue and red plots show the FWHMs for the fast-scanning (X) and slow-scanning (Y) directions. The B-scans (a) and (b) were taken from the volumes corresponding to the plots highlighted by green boxes. The used volumes are identical to those used for the MFA images in Fig. 3.

SBR plot against the number of averaged volumes N and the overall focus shifting distance D. The type of plotted dot indicates the focus shifting step Δz. The plot was produced to select optimal parameters of N, D, and Δz. The measured sample is the phantom shown in Fig. 3, and the SBR was computed by the process described in Section 3.1. The black and green plots are projections showing the relationships between the SBR and D and between the SBR and N, respectively.

En face images of the zebrafish measured with different MFA parameters at the same depth and the same lateral position. Red boxes indicate two selected regions for computing SBR. Among the four sets of parameter configurations, configuration (c) gives best contrast. This configuration was thus selected as the optimal parameter set in the present study.

Optical design schematics of the sample arm without (a) and with (b) the ETL. (c) Color photograph of the sample arm with the ETL. GS: galvanometric scanner, OBJ: objective, ETL: electrical tunable lens, and CM: collimator. Since the ETL is a refractive optical element and easy to align, switching between the two configurations without and with the ETL takes only a few minutes. (d) shows the simulation which reveals that the focus spot size becomes smaller as the focus is shifted away from the objective. The focus position is denoted by the distance from the bottom surface of the objective to the focal plane, the blue background denotes the focus shifting range D applied in the optimal protocol, and the star mark denotes the simulated spot size without ETL.