2.1.

Optical Coherence Tomography System Setup

A simple representation of an OCT system setup is shown in Fig. 1. The SD-OCT system is operated with a broadband light source (BroadLighters T-850-HP, Superlum) with a center wavelength of 860 nm, a full width at half maximum of 165 nm, and an optical power of 12.2 mW. In total, three couplers (TW850R5A2, Thorlabs) were used to set up the optical system. All three couplers are of 50:50 ratios. The input arm of coupler-1 is connected to the source, and each end of the output is connected to an individual sample arm along with its respective path length matched reference arm through coupler-2 and coupler-3. The backscattered return light in the coupler arm that is adjacent to input arm has no connection to coupler-2 and coupler-3. Coupler-2 connects with galvanometer scanner-1 (sample arm-1, which scans the top surface of the sample) and to its respective path length matched reference arm setup-1. Similarly, coupler-3 connects with galvanometer scanner-2 (sample arm-2, which scans the bottom surface of the sample) and to its respective path length matched reference setup-2. A 2-D galvanometer scanning system (GVS102, Thorlabs) is used for scanning in each sample arm setup. A 30-mm focal length lens (AC254-030-B, Thorlabs) is used for focusing the beam in both of the sample arms and in both of the reference arms. The interference backscattered lights from coupler-2 and coupler-3 are sent back to coupler-1.

Fig. 1

Schematic diagram of the bidirectional imaging modality-based SD-OCT system. BS is a broadband source; FC1, FC2, and FC3 are fiber couplers; C1, C2, and C3 are collimators; L1, L2, and L3 are lenses; M1 and M2 are mirrors; GS1 and GS2 are galvanometer scanners; LS is a leaf sample; TDG is a transmission type diffraction grating; LSC is a line scan camera; and NC means no connection.

The detector part of the system is connected to the coupling arm adjacent to the source arm of coupler-1. The detector part is comprised of collimator-3 (LB20R, Silicon Lightwave Technology), a transmission type diffraction grating (HD1800 L/mm, Wasatch Photonics), and lens-3 of 100-mm focal length (AC508-100-B, Thorlabs), which is used for focusing the diffracted rays on a 4096-pixels line scan camera (spl4096-140 km, Basler). The software for the built bidirectional SD-OCT was programmed with compute unified device architecture (CUDA) version 3.2 for Visual studio 2008, graphics card (Geforce GTX480, 700 MHz Clock rate, 480 CUDA processor, NVIDIA), and CPU (Core i7 980X Hexa cores, 3.33 GHz Clock rate, Intel). The galvanometer scanners for both sample arm-1 and sample arm-2 are driven by a personal computer (PC) with a data acquisition board (PCIe-6353, National Instruments). To attain fast data acquisition speeds, first the data acquisition thread stores the incoming 2-D signal into a buffer allocated in the random access memory in the host memory of the PC and then the signal processing thread is called. The self-repeating process continuously transfers the incoming signal to the second buffer. The frame data stored in the buffers of the host memory are copied in the device memory through the PCI express by a signal processing thread. Later, the processing divides the 480 CUDA subprocessors to further process the signal for OCT. A k-domain linearization process is carried out followed by background subtraction to remove noise in the signal. The resultant signal is Fourier transformed and log scaled to produce the 2-D OCT image. The reconstructed OCT image is then transferred back to the host memory and displayed in real-time.⁵⁸ Using LabVIEW programming, the processed 2-D OCT images are obtained with a logarithmic intensity scale. The processed 2-D OCT images and the plots derived from them were initially obtained with a linear brightness scale. The brightness scale was then varied in order to best fit the image. The lateral and axial resolutions of the system were 11.4 and $3.5\mu \mathrm{m}$ in air, respectively. A total of 500 adjacent A-scans were used to produce one 2-D image, and similarly, 500 adjacent 2-D images were processed to obtain one 3-D image. The most probable reason for the difference between the theoretical and experimental axial resolution values can be considered to be the dispersion mismatch and chromatic aberrations caused by the optical components, taking into account that the proposed system contains comparatively more optical and additional fiber components from the conventional single-sample arm-based OCT. Although, the dispersion is compensated using numerical and hardware methods, slight wavelength-dependent loss can occur due to the optical components. Consequently, the polarization mismatch between the two interferometers leads to a loss of axial resolution as well. Similarly, the imperfect Gaussian profile of the broadband source due to the three SLDs also caused a reduction in axial resolution. The built system has a frame rate of $32\text{frames}/\mathrm{s}$. Therefore, it takes about 31.25 ms to acquire one 2-D image, and for one 3-D image, i.e., 500 2-D images, it takes about 15 s.

In the proposed bidirectional imaging method, both of the reference arm powers are maintained at half that of the maximum to maintain the near saturation level of the detector. This in turn reduces the sensitivity of the individual interferometers (interferometer-1 comprised of reference arm-1 and sample arm-1; interferometer-2 comprised of reference arm-2 and sample arm-2). However, this is considered to be negligible since both of the images from the interferometers are merged to form the final OCT image, which compensates to maintain the overall sensitivity of the system.

2.2.

Alignment Method of Bidirectional Imaging Modality

Figure 2 shows a SolidWorks 3D design, illustrating the bidirectional imaging setup for the proposed system. The entire sample arm setup is mounted on a breadboard of size $300\mathrm{mm}\times 300\mathrm{mm}$. Each galvanometer scanner is mounted on a variable post holder, a translation stage with its translating micrometer head parallel to the $x$-direction, and another translation stage with its translating micrometer head parallel to the $y$-direction. The parts of sample arm-1 are indicated and named with red arrows in the SolidWorks design. Similarly, components incorporated for moving sample arm-2 are indicated with blue arrows.

Fig. 2

SolidWorks diagram representing the bidirectional imaging setup. GS1 and GS2 are galvanometer scanners; SH is a sample holder; VPH is a variable post holder in the $z$-direction; TX is a translation stage in the $x$-direction; and TY is a translation stage in the $y$-direction. Solid red and dashed blue arrows indicate sample arm-1 and sample arm-2 components, respectively

The image from sample arm-2 is inverted by moving sample arm-2 toward the sample, and the path length is matched with the respective reference arm setup-2 to obtain a clearly resolved image. Aligning both sample arms to coincide their scanning positions has vital importance to avoid errors by misinterpretation of the obtained images. To overcome this, an axial and lateral resolution target (APL-OP01 OCT phantom, Arden Photonics) is used to align the sample arms scanning beam, so that the scanning point of both sample arms coincide at the same point. The refractive index of the resolution target used is 1.45. Translation stages $x$ and $y$ were used to align the sample arms in the $x$ and $y$ directions. The variable post holders were moved accordingly to match the scanning position of both sample arms in the z-direction. By using this method, the sample arms were aligned in such a way that the scanning beams from the two sample arms are aligned with micrometer accuracy. Before and after alignment in the $x$, $y$, and $z$ directions, the respective OCT images are shown in Figs. 3(a) and 3(b). The highlighted red box regions show the differences in intensities in the depths of the 2-D image that are taken before and after alignment.

Fig. 3

Alignment of sample arms using a resolution target. (a) A 2-D cross-sectional OCT image of a lateral resolution target taken before alignment of the two sample arms. (b) The 2-D cross-sectional OCT image of a lateral resolution target taken after alignment of the two arms. The target points in the resolution target are brighter and clearly visible in (b) as compared to (a), which are highlighted in a red rectangular box.

The distance between the two sample arms can be expressed mathematically as follows:

The value of $D_{\mathrm{M}}$ for the above equation can be calculated using the following expression:

If the path lengths of the reference arms are equal, it results in high interference. This results in a broad interference line in the OCT images. To avoid this, a minimum difference has to be maintained between the optical paths. This is represented in the following equation:

The total noise ($N_{\mathrm{T}}$) occurrence in systems with multiple interferometer setups can be assessed as two types: intrainterferometric noise ($I_{\mathrm{RAIN}}$), which is the noise within an interferometer, and interinterferometric noise ($I_{\mathrm{ERIN}}$), which is the noise occurrence between the interferometers.

The former case may occur owing to phase noise between fiber connections, optical shot noise, flicker noise in FD-OCT, Gaussian white noise, and so on. We focus on the noise occurrence between the interferometers.

In the proposed system, $I_{\mathrm{ERIN}}$ will occur if the optical path lengths of the interferometers are the same. This may result in interference between the sample arms and reference arms of the interferometers. The following equation gives an overall representation of these noises:

The proposed bidirectional modality has a focal plane for each sample arm. Depending upon the sample and the depth region of interest in the sample, the foci of both sample arms can be placed as needed. The foci of both sample arms can be placed at any distance from each other as long as there is no interference between the interferometers, and it depends on the depth of focus of the individual sample arms and the fall-off of the spectrometer.

3.1.

Depth Enhancement Analysis Between the Bidirectional Imaging-Based SD-OCT Images over the Conventional Imaging Modality-Based SD-OCT Images

While scanning for the conventional SD-OCT images from the top surface of the sample, both sample arm-2 and reference arm-2 are blocked. Similarly, to see the bottom sample arm image separately, sample arm-1 and reference arm-1 are blocked. The physical thickness of the leaf was measured using a Vernier caliper. The total thickness of the leaf was $\sim 440\mu \mathrm{m}$. Figures 4(a) and 4(b) are individual sample arm images of the leaf that are taken on the adaxial and abaxial surface. Figure 4(c) is an unmerged 2-D OCT image taken with bidirectional scanning. Both of the sample arm images can be differentiated as is shown as dotted boxes in Fig. 4(c). The blue dotted box highlights the sample arm-2 image and the red dotted box highlights the sample arm-1 image. By knowing the physical thickness of the leaf measured using the Vernier caliper, the OCT images are merged accordingly. For A-scan analysis, sample regions within red, blue, and green colored boxes are used. Ten consecutive signals within these regions are normalized and the plotted graph is shown in Fig. 4(e).

Fig. 4

Individual sample arm images along with the merged images from sample arm-1 and sample arm-2. C is the cuticle; UE is the upper epidermis; M is the mesophyll; LE is the lower epidermis; SA-1 is the image from sample arm-1, SA-2 is the image from sample arm-2. (a) and (b) The respective 2-D cross-sectional images of a leaf that was individually imaged using top sample arm-1 and sample arm-2, respectively. (c) The bidirectional sample arm image without merging. (d) The bidirectional sample arm image after merging both sample arm images. (e) The normalized A-scan plots of the individual sample arms images along with the merged bidirectional sample arm image. Rectangular boxes in red, blue, and green color in figures (a), (b), and, (d) respectively, are the regions used for A-scan analysis.

Sensitivity is a function of change in refractive index and the change in optical path difference between sample and reference arm. As we know, the PSF has depth-dependent sensitivity roll off with optical path difference in the case of a mirror as a sample.³⁸ The sensitivity profile in OCT image is dependent on the change in refractive index within the sample. The points with high-refractive index change have high sensitivity.

Figures 5(a) and 5(b) are the cross-sectional images of 7 Scotch tapes stacked one above another. The total thickness measured using a Vernier caliper was $450\mu \mathrm{m}$. In Fig. 5(a), the middle and lower layers of the Scotch tapes are not clear. The cross-sectional image obtained using the bidirectional imaging setup gives more depth information and the layers of the sample can be seen clearly. Manually measured sample thickness is used for the alignment of the inverted sample arm-2 image by the aforementioned alignment procedure.

Fig. 5

Depth enhancement analysis in stacked Scotch tape and silicon sheets; BSS is the bottom surface of the silicon sheet; BSN is the bottom surface of the needle; TDEA is the total depth enhancement achieved; DLI is the difference in layer intensities. (a) and (b) The respective 2-D cross-sectional images of 7 Scotch tapes obtained with the conventional imaging modality setup and bidirectional imaging modality setup of an SD-OCT system. The blue and red box regions were used for A-scan analysis. (d) The normalized A-scan plot of (a) and (b). (c) and (e) The respective 2-D cross-sectional OCT images of a silicon sheet impaled by two needles at middle, obtained using the conventional and bidirectional SD-OCT systems.

Figures 5(c) and 5(e) are 2-D cross-sectional images of a silicon sheet with a thickness of 1.7 mm. The silicon sheet was impaled by two needles. The refractive index of the silicon sheet is $\sim 1.45$. A visible top layer and a marginally visible bottom layer of the silicon sheet can be seen in the conventional SD-OCT image. Furthermore, the clear visibility of both top and bottom layers of the sample using a dual-sample arm shows accurate depth enhancement. A-scan analysis plots are shown in Fig. 5(d). A set of 10 consecutive A-scans of each 2-D image were used for analyses that are highlighted in the blue and red box regions Figs. 5(a) and 5(b). The exact same area is chosen for comparative analysis. From the normalized A-scan plot, it can be seen that a total depth enhancement of $200\mu \mathrm{m}$ is achieved in the Scotch tape sample by using the bidirectional/dual-scan imaging method over the conventional single scan image. In A-scan analysis plot, the total depth enhancement achieved (TDEA) is calculated by taking the start point of the limit at 15% or less of the intensity of the maximum peak and after that no further occurrence of higher intensity is observed. The end point is taken where a significant increase in intensity can be observed by the second sample arm.

3.2.

Persimmon Leaf Image Comparison Between the Bidirectional Imaging Modality and the Conventional Imaging Modality

For image comparison between the proposed method and the conventional imaging modality, a persimmon leaf sample was utilized. Scanning was performed on the same position of the leaf using the proposed and conventional setups.

The scanning range used for both setups was $3\mathrm{mm}\times 3\mathrm{mm}$. The obtained 500 adjacent B-scan images were processed to make the 3-D images. Figure 6 images were obtained using the dual-sample arm-based SD-OCT system. Figure 6(a) shows the 3-D image and Fig. 6(c) shows the cross-sectional 2-D image. Distinguishable layers of the cuticle, upper and lower epidermis, and mesophyll are marked using red arrows in Fig. 6(c). Figure 6(b) is the orthosliced view from all three directions of axial, coronal, and sagittal. Figure 6(d) is the enlarged view of the orthoslice in the coronal direction. The highlighted red box region shows the clear and detailed structure of the persimmon leaf. All orthoslices were at the middle position of the 3-D image.

Fig. 6

Bidirectional SD-OCT images of the persimmon leaf. C is the cuticle; UE is the upper epidermis; M is the mesophyll; LE is the lower epidermis. (a) A 3-D image of the persimmon leaf. (b) The orthoslice of the 3-D image from all three directions (axial, coronal, and sagittal). (c) The cross-sectional 2-D image taken using the bidirectional SD-OCT. (d) An enlarged view of the orthosliced image from the coronal direction.

Figure 7 was obtained using the conventional imaging modality (single-sample arm)-based SD-OCT system. Figure 7(a) shows the 3-D image, and Fig. 7(c) shows the cross-sectional 2-D image. Not all layers of the persimmon leaf are visible, unlike in Fig. 6(c). Only the cuticle in the top surface of the leaf, upper epidermis, and most of the mesophyll is visible in the obtained cross-sectional 2-D image using a conventional SD-OCT setup. Figure 7(b) is the orthosliced view from all three directions of axial, coronal, and sagittal. The orthoslice was at the same position as in Fig. 6(b). Figure 7(d) is the enlarged image of the orthoslice in the coronal direction, and the highlighted blue box region shows reduced intensity and the missing structural details in the mesophyll layer as compared to Fig. 6(d).

Fig. 7

Conventional single-sample arm-based SD-OCT images of the persimmon leaf. C is the cuticle; UE is the upper epidermis; and M is the mesophyll. (a) A 3-D image of the persimmon leaf. (b) The orthoslice of the 3-D image from all three directions [axial, coronal, sagittal are at the same position as (b)]. (c) The cross-sectional 2-D image taken using the conventional SD-OCT. (d) The enlarged view of the orthosliced image in the coronal direction and the point of orthoslice is also the same as the image taken in Fig. 6(d).

3.3.

Comparison of the A-Scan Analysis Between the Bidirectional and the Conventional SD-OCT Images

For normalized A-scan analysis, the same location of the persimmon leaf is imaged using the bidirectional and conventional imaging modalities. In the obtained cross-sectional 2-D images, the exact same area is selected for comparative analysis. A set of 10 consecutive A-scans of each 2-D image were used for analysis. The red box region in Fig. 8(a) and blue box region in Fig. 8(b) were used for averaged A-scan analysis. The position and total area used for analysis is approximately the same in both Figs. 8(a) and 8(b).

Fig. 8

Comparison of A-scan analysis of the bidirectional/dual-sample arm and the conventional single-sample arm-based SD-OCT images. C is the cuticle; UE is the upper epidermis; M is the mesophyll; LE is the lower epidermis; DLI is the difference in layer intensities; TDEA is the total depth enhancement achieved. (a) A 2-D cross-sectional image of the persimmon leaf taken using the bidirectional imaging SD-OCT system. The red rectangular box region indicates the area used for A-scan analysis. (b) A 2-D cross-sectional image taken in the same position of the same leaf using the conventional imaging SD-OCT system. The blue rectangular box region indicates the area used for A-scan analysis. (c) The normalized A-scan analysis plot done on (a) and (b).

From the obtained normalized A-scan analysis, it is clearly seen that the intensity of the peaks increases, thus the intensity fall-off with depth can be compensated by using the bidirectional imaging method. The intensity increase in layers is indicated with brown arrows in the A-scan plot. The TDEA using bidirectional imaging is about $80\mu \mathrm{m}$ for this particular sample, which is indicated with dotted black lines in Fig. 8(c) (A-scan plot). Therefore, it is confirmed that more details and structures of the leaf layers could be identified using the bidirectional imaging method.