Space-division Multiplexing Optical Coherence Tomography References and Links Multi-channel Fourier Domain Oct System with Superior Lateral Resolution for Biomedical Applications, "

High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT). Here, we demonstrate a space-division multiplexing (SDM) technology that translates long coherence length of a commercially available wavelength tunable laser into high OCT imaging speed. We achieved an effective 800,000 A-scans/s imaging speed using a 100,000 Hz tunable vertical cavity surface-emitting laser (VCSEL). A sensitivity of 94.6 dB and a roll-off of < 2 dB over ~30 mm imaging depth were measured from a single channel in the prototype SDM-OCT system. An axial resolution of ~11 μm in air (or ~8.3 μm in tissue) was achieved throughout the entire depth range. An in vivo, 3D SDM-OCT volume of an entire Drosophila larva consisting of 400 x 605 A-scans was acquired in 0.37 seconds. Synchronized cross-sectional OCT imaging of three different segments of a beating Drosophila larva heart is demonstrated. The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling. SDM-OCT also preserves image resolution and allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications. High speed spectral domain optical coherence tomography for retinal imaging at 500,000 A-lines per second, " Biomed. A. Leitgeb, " Heartbeat phase coherent Doppler optical coherence tomography for measuring pulsatile ocular blood flow, " OCT: high quality 3D imaging at 20 million A-scans and 4.5 GVoxels per second, " Opt. Domain Mode Locking (FDML): A new laser operating regime and applications for optical coherence tomography, " Opt. Buffered Fourier domain mode locking: unidirectional swept laser sources for optical coherence tomography imaging at 370,000 lines/s, " Opt. Sensitivity advantage of swept source and Fourier domain optical coherence tomography, " Opt. Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomography, " Opt. Performance of fourier domain vs. time domain optical coherence tomography, " Opt. " Ultrahigh speed 1050nm swept source/Fourier domain OCT retinal and anterior segment imaging at 100,000 to 400,000 axial scans per second, " Opt. " Visualization of microvasculature by dual-beam phase-resolved Doppler optical coherence tomography, " Opt. domain optical coherence tomography using optical demultiplexers imaging at 60,000,000 lines/s, " Opt. Lett. Spectral domain optical coherence tomography of multi-MHz A-scan rates at 1310 nm range and real-time 4D-display up to 41 volumes/second, " Biomed. Opt. Full range complex spectral optical coherence tomography technique in eye imaging, " Opt. High speed full range complex spectral domain optical coherence …


Introduction
High speed, high resolution and high sensitivity are desirable for optical coherence tomography (OCT), an emerging "optical biopsy" technique with resolutions approaching that of histopathology [1][2][3].OCT imaging speed, characterized by the number of axial scans per second (A-scans/s), is limited by the line rate of line-scan cameras for spectral-domain OCT, or by the laser sweep rate for swept-source OCT, respectively.Commercially available line-scan cameras and tunable lasers operate at about 20,000 -300,000 A-scans/s [4][5][6].A number of approaches have been pursued to improve OCT imaging speed.The first approach is to develop high-speed line-scan cameras or high sweep rate tunable lasers.A record sweep rate of 5.2 MHz was demonstrated [7] using the Fourier-domain mode-locking (FDML) laser technology [8,9].However, as the axial scan rate increases, the dwell time at each imaging point decreases proportionally, resulting in a significant reduction in image sensitivity [10][11][12].In general, the number of sampling points for each axial scan decreases as the axial scan rate increases.For tunable lasers, the wavelength sweep range is often compromised when the laser sweep rate increases, reducing the axial image resolution for OCT [7].The second approach is to use parallel detection channels to improve OCT imaging speed [7,[13][14][15][16].However, the scalability of this approach is poor due to unfavorable cost scaling.The high system complexity and the associated cost have prohibited wide utilization of multi-beam OCT systems using parallel detection channels to date.
Extended imaging range with minimal sensitivity roll-off can be highly beneficial for ranging applications and for imaging samples with large variation of surface topography, such as whole eye imaging, endoscopic and cardiovascular imaging.Several techniques have been developed to increase the axial imaging range of Fourier-domain OCT.These include phase shifting using a piezo translator [17] or an electro-optic phase modulator [18] on the reference arm, heterodyne detection [19,20], polarization encoding [21], BM-mode scanning [22], dispersion encoding [23], coherence revival [24], and depth-multiplexing [25].Depthmultiplexing techniques have also been utilized in simultaneous imaging of anterior segment and retina [26], and in polarization-sensitive OCT systems for simultaneously detection of orthogonal polarization states using electric-optic or acousto-optic devices [27] or passive optical delay units [28,29].A commercial OCT system, i.e. the VivoSight OCT scanner from Michelson Diagnostics Ltd., utilizes multiple beams focused at different depths on samples to obtain extended depth of focus [30].
With the advancements in swept-source technology, there has been great interests in developing OCT light sources with extended coherence length for obtaining longer imaging range [13,[31][32][33][34][35][36][37][38].Recently developed wavelength tunable vertical cavity surface-emitting lasers (VCSEL) achieved coherence length of over 50 mm [36,38].Due to high scattering in biological tissues and limited depth of focus in order to achieve micron-scale transverse resolutions, OCT imaging depth is generally within 1-2 mm from the tissue surface.This provides a unique opportunity to further increase OCT imaging speed by utilizing the long coherence length property of newly developed tunable lasers.
Here, we demonstrate a space-division multiplexing (SDM) technology that translates long coherence length of a commercially available tunable laser into high OCT imaging speed.We achieved an effective 800,000 A-scans/s imaging speed using a commercial 100,000 Hz tunable VCSEL light source [31,[35][36][37][38][39].The SDM technology provides a new orthogonal dimension for further speed improvement for OCT with favorable cost scaling.The SDM-OCT technology we present here can achieve an order of magnitude or more improvement in imaging speed without compromising axial image resolution.SDM-OCT also allows synchronized cross-sectional and three-dimensional (3D) imaging of biological samples, enabling new biomedical applications.Returned signal from different sample locations were combined at the PLC splitter and interfered with light from the reference arm.A single detector and a high speed data acquisition card were utilized to record interference signal from all beams simultaneously.Since each beam was optically delayed, signal from different locations was presented at different frequency range [i.e.imaging depth, Fig. 1(c)].Since all the beams were scanned together, synchronized imaging from multiple sample locations was enabled.This technique allows parallel imaging from multiple sample locations and therefore improves OCT axial scan rate by a factor equal to the number of beams used simultaneously.

Space-division multiplexing OCT system
A commercial VCSEL tunable laser (SL1310V1, Thorlabs Inc.) with a center wavelength of 1310 nm was used for the prototype SDM-OCT system.The laser has a sweep rate of 100,000 Hz, a duty cycle of ~65%, a tuning range of 100 nm, a coherence length of over 50 mm, and output power of 37 mW.A 95/5 optical coupler (AC Photonics, Inc.) was connected to the laser output.Five percent of the light was directed to a Mach-Zender interferometer (MZI), which had an optical path length difference of 60 mm in air.The remaining 95% of the light was used for OCT imaging.A 90/10 optical coupler provided 10% of the light to the reference arm and 90% of the light to the sample arm.Input light to the sample arm was split into 8 fibers using a PLC splitter (PLC Connections, LLC.).Output fibers from the PLC splitter were custom arranged in a one-dimensional (1D) array [1x8, Fig. 1(b)], with a length difference of 2.5 mm between each optical fiber.Spacing was ~300 μm between each fiber in the 1D array and ~370 μm after the spots were projected on samples.Surface of the fiber array was angle polished at 8 degree to minimize light reflection.An f = 30mm collimator was used in the sample arm, resulting in ~8 mm beam diameters.Light from different fibers were focused onto different spots on the sample, which were synchronously scanned by a pair of galvanometers (6215H with a pair of 10 mm mirrors, Cambridge Technology, Inc.).A 5X objective lens (Mitutoyo, 5X NIR) was utilized to provide ~11 μm transverse resolutions.Each spot had ~2 mW power on sample.Light intensity variation among all the spots was less than 1 dB.Optical circulators (AC Photonics, Inc.) were utilized on both the sample and reference arms to route reflected signal from both arms to interfere at a 50/50 coupler.Broadband balanced detectors (1.6 GHz, PDB480C-AC, Thorlabs Inc.) were used to detect interference signal from both OCT and MZI systems.Outputs of the balanced detectors were digitally acquired simultaneously using a high speed data acquisition card (ATS9360, without on-board memory, Alazar Technologies Inc.) at 1.2 GS/s.Data from both channels (8320 points for each channel per sweep) was streamed to the computer memory continuously through a PCIe 8x port.The entire imaging range of the prototype system was about 35 mm in air.

Drosophila melanogaster larvae preparation
Drosophila melanogaster is a widely used model system for genetic and developmental biology studies due to the ease in culturing and its short lifecycles.In vivo SDM-OCT images were obtained from third instar Drosophila larvae.At this stage, the heart tube is located at the dorsal midline, spanning segments T2 (anterior end) to A8 (posterior end), being attached to its dorsal epidermis by seven pairs of alary muscles [40].The larva was gently mounted onto a black clip board using a double-sided tape with its dorsal side facing upwards for imaging.No anesthesia was used to immobilize the Drosophila larvae.

In vivo imaging and signal processing
In order to image an entire Drosophila larva, all 8 beams in the SDM-OCT system were illuminated along the larva dorsal midline simultaneously.A 3D data set, consisting 400 x 80 laser sweeps with each beam scanned a 1.1 mm x 0.4 mm range, was acquired in 0.37 s.The MZI signal acquired simultaneously with the OCT signal was used for phase calibration of each laser sweep [36].Images from the 8 beams were segmented from different depth range and digitally combined to form a volumetric data set consisting 400 x 605 A-scans covering the entire larva (1.1 mm x 3.0 mm range) using a custom written Matlab script.There were about 5 B-scans overlapping for adjacent 3D volumes from the 8 beams.To obtain M-mode imaging from the larva heart, 400 B-scans with each containing 400 A-scans over 250 μm range around the heart tube were acquired in 2 s (~217 frames per second).Images from all 8 beams were collected simultaneously, although only images from three different heart segments were digitally combined and presented side-by-side for comparison.Functional information of the heart was analyzed following established methods [41,42].Matlab (Mathwork, Inc.) was used to process the data.ImageJ (NIH) and Amira (VSG, Inc.) were used to generate videos and render images for presentation.

Point-spread-functions (PSFs) and sensitivity roll-off measurements
Figure 2 shows point-spread-functions (PSFs) and sensitivity roll-off measurements of the prototype system.A pin-hole was used on the sample arm to allow only the center beam to pass through for these measurements.A 94.6 dB sensitivity was measured using a calibrated reflector (−43.4 dB) and a sensitivity roll-off of < 2 dB was observed at ~30 mm imaging depth.The axial resolution was measured to be ~11 μm in air (or ~8.3 μm in tissue) throughout the entire depth range.

SDM-OCT imaging of Scotch tapes
We first performed SDM-OCT imaging on a roll of Scotch tape [Fig.3] in order to demonstrate the image quality.As shown in Fig. 3(a), tape images seen from different depth corresponded to images obtained from different beams labeled as 1 to 8. Figure 3(b) shows magnified images of the tape at locations 1 through 8. Relatively uniform image intensity was observed from all the beams.Even at the deepest imaging depth (beam #8), layers of the tape were clearly visible.Since the laser was operated at 100,000 Hz, an effective 800,000 Ascans/s was achieved combining signals from all 8 beams.).An effective OCT imaging speed of 800,000 A-scans/s was achieved combining all 8 beams.Scale bars, 500 μm.

3D SDM-OCT imaging of Drosophila larva
We further demonstrated in vivo 3D SDM-OCT imaging of a Drosophila larva [Fig.4].The entire 3D data set [400 x 605 A-scans, Fig. 4(a), and Media 1], assembled using images from all 8 beams, was acquired in 0.37 second.Figures 4(b) and 4(c) show cross-sectional and enface images of the larva, respectively.The heart tube and the trachea structures were clearly observed.

Synchronized imaging of drosophila larva heart using SDM-OCT
To demonstrate synchronized imaging capability, we performed M-mode B-scan imaging (217 frames per second) over three segments of another larva heart, estimated to be the A7, A6, and A5 segments.A representative cross-sectional image showing three segments of the heart tube is shown in Fig. 5.A video demonstrating synchronized beating of the segments is provided as Media 2. We further quantified cardiac functions of the larva using a method we developed earlier [41,42].In addition to quantifying heart rate (372 beats per minute), end systolic (36 μm for A7, 32 μm for A6, and 11 μm for A5) and diastolic dimensions (60 μm for A7, 74 μm for A6, and 58 μm for A5) and fractional shortening (42% for A7, 56% for A6, and 80% for A5), we also observed a delay between the dilation and contraction in segment A6 compared to A7 (14 ms), and segment A5 compared to A6 [69 ms, Fig. 6].This delay suggested the contraction of the heart tube was initialized in the posterior segment and propagated toward the anterior segment.This finding is consistent with previous literatures on Drosophila larvae heart development [43].laser tunable range was not changed.Meanwhile, the dwell time at each imaging spot was longer using a low tunable rate laser.More photons were reflected from the sample and more data sampling points were recorded within the dwell time for SDM-OCT.This was in contrast to the standard approach, where the dwell time and the number of sampling points for each axial scan were reduced in order to achieve high axial scan rate.For example, assuming the same power level at the imaging spot, a single-spot system running at 800 kHz would have 8 times less photons reflected back from the sample within the dwell time, compared to an 8 x 100 kHz SDM-OCT system.This corresponds to ~9 dB sensitivity reduction for the singlespot OCT system.In our current prototype system, however, the PLC splitter had a ~10 dB insertion loss.This was not an issue in the forward direction, where the input light was evenly split into the 8 beams.However, when combining reflected light from the sample, the 10 dB insertion loss resulted in a significant reduction in sensitivity.As a result, we achieved only ~95 dB sensitivity over the entire imaging range when ~2 mW light was shine on the sample at each imaging spot.This was about 11 dB lower compared to the shot-noise-limited sensitivity.In the future, alternative designs bypassing the PLC splitter in the return path may be utilized to further improve imaging sensitivity.
With the SDM technology, the bottleneck for further improving swept-source OCT imaging speed is no longer the sweep rate of tunable lasers.Instead, high speed data acquisition and high throughput data transfer are greatly desired.The data acquisition card in our prototype system supports a 12-bit 1.8 GS/s sampling rate.However, due to limited data transfer rate, we could only sample the OCT and MZI channels simultaneously at 1.2 GS/s.Further improvement in data acquisition and transfer rate, bandwidth of the detectors, coherence length of the light source, and number of fiber channels will further scale up OCT axial scan rate linearly with minimum additional cost.Since the input light is split into multiple beams, a powerful light source is also desirable.

Conclusions
In summary, we demonstrated a space-division multiplexing technique for OCT.SDM-OCT achieves significant improvement in axial scan rate and preserves axial imaging resolutions using a passive optical component and a single detection channel.This technique enables synchronized cross-sectional and 3D imaging of biological samples, and provides a new cost favorable approach to further improve OCT axial scan rate.SDM-OCT may be applied to a wide range of structural and functional OCT applications, including but not limited to ophthalmology, cardiovascular imaging, endoscopic imaging, cancer imaging and dental applications.

Figure 1
Figure1demonstrates the concept for SDM-OCT.The key of the technology is to create multiple imaging beams to illuminate the sample simultaneously, while having different optical delays for each beam [see the red rectangular region in Fig.1(a).For simplicity, only 4 beams are shown in the figure].This was realized by using a planar lightwave circuit (PLC) splitter and a fiber array [Fig.1(b)].Returned signal from different sample locations were combined at the PLC splitter and interfered with light from the reference arm.A single detector and a high speed data acquisition card were utilized to record interference signal from all beams simultaneously.Since each beam was optically delayed, signal from different locations was presented at different frequency range [i.e.imaging depth, Fig.1(c)].Since all the beams were scanned together, synchronized imaging from multiple sample locations was enabled.This technique allows parallel imaging from multiple sample locations and therefore improves OCT axial scan rate by a factor equal to the number of beams used simultaneously.A commercial VCSEL tunable laser (SL1310V1, Thorlabs Inc.) with a center wavelength of 1310 nm was used for the prototype SDM-OCT system.The laser has a sweep rate of 100,000 Hz, a duty cycle of ~65%, a tuning range of 100 nm, a coherence length of over 50 mm, and output power of 37 mW.A 95/5 optical coupler (AC Photonics, Inc.) was connected to the laser output.Five percent of the light was directed to a Mach-Zender interferometer (MZI), which had an optical path length difference of 60 mm in air.The remaining 95% of the light was used for OCT imaging.A 90/10 optical coupler provided 10% of the light to the reference arm and 90% of the light to the sample arm.Input light to the sample arm was split into 8 fibers using a PLC splitter (PLC Connections, LLC.).Output fibers from the PLC splitter were custom arranged in a one-dimensional (1D) array [1x8, Fig.1(b)], with a length difference of 2.5 mm between each optical fiber.Spacing was ~300 μm between each fiber in the 1D array and ~370 μm after the spots were projected on samples.Surface of the fiber array was angle polished at 8 degree to minimize light reflection.An f = 30mm collimator was used in the sample arm, resulting in ~8 mm beam diameters.Light from different fibers were focused onto different spots on the sample, which were synchronously scanned by a pair of galvanometers (6215H with a pair of 10 mm mirrors, Cambridge Technology, Inc.).A 5X objective lens (Mitutoyo, 5X NIR) was utilized to provide ~11 μm transverse resolutions.Each spot had ~2 mW power on sample.Light intensity variation among all the spots was less than 1 dB.Optical circulators (AC Photonics, Inc.) were utilized on both the sample and

Fig. 1 .
Fig. 1.Concept for SDM-OCT.a).Schematic diagram of the prototype SDM-OCT system.The key of the technology is to create multiple illumination beams on the sample simultaneously, while having different optical delays for each beam (see the red rectangular region).A single detection channel was used to collect signals from all beams simultaneously.b).A 1x8 fiber array with 300 μm spacing between individual fibers was used in the prototype system.c).Each beam was optically delayed.Signals from different beams (different sample locations) were presented at different frequency range (i.e.imaging depth).For simplicity, only 4 beams are shown.

Fig. 2 .
Fig. 2. Point spread functions (PSFs) and sensitivity roll-off measurements of the SDM-OCT imaging system.a). and c).Log and linear scale plot of the PSFs throughout the entire imaging depth.b). and d).Log and linear PSFs at ~30 mm imaging depth.

Fig. 3 .
Fig. 3. SDM-OCT imaging of a roll of Scotch tape.a).Representative B-scan image of the tape.An imaging depth of 35 mm in air was obtained.Tape images seen from different depth (labeled as 1 to 8) corresponded to images obtained from different beams.b).Magnified view of tape images at locations 1 to 8. Layers of the tape were clearly visible even at the deepest imaging range (beam #8).An effective OCT imaging speed of 800,000 A-scans/s was achieved combining all 8 beams.Scale bars, 500 μm.

Fig. 4 .
Fig. 4. In-vivo 3D SDM-OCT imaging of a Drosophila melanogaster larva.a).3D volumetric data of a Drosophila larva (400 x 605 A-scans, Media 1) was obtained in 0.37 second by combining images from all 8 beams.b).Cross-sectional and c).en-face images of the larva, showing the heart tube and the trachea structures clearly.Scale bars, 250 μm.

Fig. 6 .
Fig.6.M-mode images of the three heart segments (A7 -A5) and measurements of transverse heart chamber dimensions.Time delays between dilation and contraction in the three segments suggested that the contraction of the heart tube was initialized in the posterior segment and propagated toward the anterior segment.Scale bars, Vertical, 100 μm, horizontal, 0.2 s.