Special Section on Selected Topics in Biophotonics: Optical Coherence Tomography and Biomolecular Imaging with Coherent Raman Scattering Microscopy

Optical coherence tomography today: speed, contrast, and multimodality

[+] Author Affiliations
Wolfgang Drexler

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Mengyang Liu

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Abhishek Kumar

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Tschackad Kamali

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Angelika Unterhuber

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

Rainer A. Leitgeb

Medical University of Vienna, Center for Medical Physics and Biomedical Engineering, Waehringer Guertel 18-20, A-1090 Vienna, Austria

J. Biomed. Opt. 19(7), 071412 (Jul 31, 2014). doi:10.1117/1.JBO.19.7.071412
History: Received February 11, 2014; Revised April 4, 2014; Accepted April 25, 2014
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Abstract.  In the last 25 years, optical coherence tomography (OCT) has advanced to be one of the most innovative and most successful translational optical imaging techniques, achieving substantial economic impact as well as clinical acceptance. This is largely owing to the resolution improvements by a factor of 10 to the submicron regime and to the imaging speed increase by more than half a million times to more than 5 million A-scans per second, with the latter one accomplished by the state-of-the-art swept source laser technologies that are reviewed in this article. In addition, parallelization of OCT detection, such as line-field and full-field OCT, has shortened the acquisition time even further by establishing quasi-akinetic scanning. Besides the technical improvements, several functional and contrast-enhancing OCT applications have been investigated, among which the label-free angiography shows great potential for future studies. Finally, various multimodal imaging modalities with OCT incorporated are reviewed, in that these multimodal implementations can synergistically compensate for the fundamental limitations of OCT when it is used alone.

Optical coherence tomography (OCT) is one of the most innovative and rapidly emerging optical imaging modalities in the last decades14 because it is capable of perfectly noninvasively exploiting the wealth of morphologic and functional tissue information in the first few millimeters of organs. Since the beginning of OCT in the late 1980s and beginning of the 1990s,57 more than 50 OCT companies have been created, more than a hundred research groups are involved in OCT, over a thousand OCT patents have been granted, and more than ten thousand research articles have been published—mostly in ophthalmology, followed by technology-related journals and cardiovascular publications (http://www.octnews.org/). In ophthalmic diagnosis especially, OCT is the fastest adopted imaging technology in the history of ophthalmology. While 108 million x-rays, 30 million single-photon emission computed tomographys (CT), positron emission tomographys, and CTs, and 26 million magnetic resonance (MR) imaging examinations have been performed in 2010, 32 million ophthalmic OCT scans have already been conducted (http://www.octnews.org/). In >110 years of x-ray imaging development, the bionizing radiation dose has been reduced by 1500 times; imaging speed is 257,000 faster; its contrast significantly increased and the images are much less blurry.8 In <20 years of OCT development, on the contrary, the axial resolution has been improved by >10 times; the imaging speed is increased by more than half a million times; image contrast is greatly enhanced and many functional extensions of OCT have been developed.

The original idea of OCT was to enable noninvasive optical biopsy, i.e., the in situ imaging of tissue microstructure with a resolution approaching that of histology without the need for tissue excision and processing.9 Thus, we have the following prerequisites: (1) visualization of subcellular morphology (i.e., ultrahigh isotropic resolution), (2) three-dimensional imaging (resulting in the need for ultrahigh imaging speed), (3) sufficient (tissue) contrast, (4) molecular, biochemical sensitivity (i.e., sensitive to absorption of diagnostically relevant endogenous or exogenous chromophores), (5) localized (quantitative) functional tissue information, and (6) sufficient (diagnostically relevant) tissue penetration. Axial resolution improvement (prerequisite 1) has been the key technological milestone in the history of OCT in its first decade,1014 which has been achieved by using ultra-broadband sources. OCT imaging speed improvements (prerequisite 2) evolved in its second decade, which were accomplished by Fourier domain (FD)/spectral domain (SD)1518 and swept source (SS) OCT.1923 While in FD/SD OCT speed is mainly determined by the readout time of the camera in a spectrometer, the wavelength-tuning speed of swept sources is the decisive factor in SS OCT. In the past 5 to 10 years, different swept source technologies have emerged to significantly improve imaging speed in (commercial) OCT systems—especially the ones using wavelengths >1μm. In the present paper, the state-of-the-art (ultra)high-speed SS technologies are reviewed and compared (cf. Sec. 2.) and an outlook of even further improving OCT imaging speed via parallelization (line- and full-field OCT) of OCT detection is presented (cf. Sec. 3). OCT contrast enhancement (prerequisite 3) has been accomplished by introducing polarization-sensitive OCT,2428 phase-sensitive OCT,2933 optical coherence elastography,3439 spectroscopic low coherence interferometry,4043 elastic scattering spectroscopy,31,4446 and nonlinear interferometric vibrational imaging (NIVI), as well as employing endogenous or exogenous contrast.4752 In this paper, a recently developed contrast improvement for OCT, named label-free optical angiography, is reviewed (cf. Sec. 4). Some of the above-mentioned contrast enhancement OCT extensions are targeted to enable OCT molecular and biochemical sensitivity (prerequisite 4) as well as localized (quantitative) functional tissue information (prerequisite 5), with the most successful one being Doppler OCT. Though having been improved in many aspects, OCT so far is still not sensitive enough to provide absorption contrast or biochemical and molecular information. Therefore, multimodal optical imaging modalities with OCT incorporated have become a hot research topic. In this paper, the following combinations are reviewed: OCT with multiphoton tomography (MPT, for subcellular resolution); OCT with nonlinear microscopy (for label-free molecular tissue information, cf. Sec. 5.1); and OCT with photoacoustic imaging (for enhanced absorption sensitivity and penetration depth—prerequisite 6, cf. Sec. 5.2). Previous reviews of OCT focused on advanced screening in the fields of primary care,53 microscopy,54 rapid tissue screening,55 phantoms,56 high speed,57 and translational research,58 while recent application-specific OCT reviews covered ophthalmology,5961 cardiology,6266 dermatology,6770 novel applications in pulmonary medicine,71 cancer,72 and optical coherence elastography,73 as well as OCT post- and signal processing.74,75 This review focuses on high-speed technology (SS light technology and parallelization of OCT detection), label-free angiography, and multimodal OCT.

Despite the boom of new SS technologies in recent years, it is noteworthy that none of them is at the moment even close in maturity and reliability to that of the superluminescent laser diodes (SLDs) that have been already employed in (commercial) OCT systems. Nevertheless, swept sources seem to be the OCT technology of the future since they have the potential to offer significantly higher scanning speeds, extended depth range with significantly reduced sensitivity roll-off, reduced fringe washout from sample motion or rapid transverse scanning, and improved light detection efficiency due to dual balanced detection. SS OCT avoids the need for spectrometers with line scan cameras, but requires a high-speed, narrow line width swept source. Spectrometers have limited spectral resolution due to the grating resolving power, beam spot size, and finite pixel dimensions of the line scan cameras. Narrow line width swept source enables spectral resolution in SS OCT, which can be much higher than that of SD OCT. For OCT applications using wavelengths >1μm, SS OCT seems to be the most promising technology of choice because indium gallium arsenide (InGaAs) line cameras are limited in speed and number of pixels, and are more expensive than silicon line cameras. SD OCT suffers from significantly more SNR roll-off with scanning depth than that achievable by using narrow line width swept sources. The seven most important swept laser technologies (from six companies; cf. Table 1) are reviewed and compared in terms of their potential for (commercial) OCT systems. Only those technologies that have been commercialized or are on the verge of commercialization have been reviewed. Novel emerging scientific approaches for swept sources are not considered in this paper.

Table Grahic Jump Location
Table 1Comparison of state-of-the-art swept source technology.
Axsun Technologies Inc., Massachusetts (“a Volcano Company”)

The laser engine consists of a swept laser module, control electronics, k-clock, balanced receiver, and data acquisition board, which samples on k-clock transitions (information in this section is from www.axsun.com). More specifically, it contains a reflective microelectromechanical system (MEMS) tunable Fabry-Perot filter, a broadband gain chip, and a fiber reflector that forms the other end of the laser cavity and serves as the output coupler. Filter tuning is accomplished by changing the drive voltage on the MEMS filter. Fiber extension brings the equivalent air length of the cavity to 104 mm such that there are a handful of laser cavity modes underneath the filter at all times. The fact that this external cavity laser operates with a cluster of modes, rather than a single mode, leads to a coherence length that is an order of magnitude or more smaller than single-mode semiconductor lasers. Multimode operation can also increase relative intensity noise (RIN) though. The 100-kHz laser operates with two pulses traveling in the cavity at once separated by half the cavity round-trip time. With longer path mismatches, pulses can interfere with their neighbors, leading to the coherence revival phenomenon. This external cavity laser uses a digital k-clock, which is derived from a fiber-based Mach-Zehnder interferometer. Main envisaged innovations include doubling the scanning speed to 200 kHz and increasing coherence length by adjusting the filter bandwidth.

Hamamatsu Photonics K.K. (NTT-Advance Technology Corp.), Tokyo, Japan

A high-speed KTa1−xNbxO3 (KTN) light deflector is used in an external-cavity laser (Littman-Metcalf cavity configuration) that has no moving parts (mechanical free high-speed operation) (information in this section is from www.ntt-at.com). It deflects light using an electro-optic effect. KTN has a very large electro-optic effect (20× higher than lithium niobate), which changes a refractive index by an applied voltage and bends the path of a light beam in a direction. The KTN crystal is precharged by applying ±500V dc for 10 s and then scans the laser wavelength by applying a ±400V sinusoidal voltage to the crystal. However, KTN simultaneously exhibits the characteristics of a cylindrical convex lens because of the trapped electrons. This convex lens is compensated with a cylindrical concave lens because the lens power inside the cavity degrades the instantaneous line width of the laser. At the moment, the scanning wavelength range is 80nm with 20 mW output power and up to 200 kHz (sinusoidal) sweep speed with a ±400V deflector driving voltage at 1310 nm. The duty ratio changes from 50:50 to 70:30—at the moment, 200 kHz drive signal with one-way imaging—enabling a 50% duty cycle. Main envisaged innovations include 100 nm sweep width, 20 mW optical output power at 1310 nm with 200 kHz sweep rate and 8 mm coherence length—equivalent to 4 mm scanning depth (6dB signal roll-off), standard deviation of timing jitters between adjacent A-lines: <200ps, which corresponds to a phase difference of <0.05rad at a path difference of 1.5 mm of a Michelson interferometer.

Insight Photonics Solutions Inc., Lafayette, Colorado

Sweep flexibility of the akinetic laser is purely software-driven. The all-semiconductor laser’s distributed Bragg reflector-like structure increases the finesse of the cavity by a factor of approximately nine times, resulting in a narrow line width (information in this section is from www.sweptlaser.com). The small dimensions of the laser cavity and the fact that the entire cavity is on a single rigid structure substantially reduce cavity length variation. Superior line width and spectral performance are natural outcomes of the very short waveguide design. Experimental data show that moving from any wavelength to an adjacent wavelength with the all-semiconductor laser takes 2.5ns. Because the duty cycle is software-controlled, it is adjustable and, hence, can be set to almost any unidirectional value from 5 to 95%. The all-semiconductor laser is inherently linear and does not require an external optical k-clock. The laser self-generates an internal electronic k-clock. The laser forces the wavelength to be correct at each of the evenly spaced clock transitions. The all-semiconductor laser does not need the extra cost of parts and integration of an optical k-clock, and eliminates the challenges frequently associated with the nonuniform triggering that can occur with external optical k-clocks. The result is direct triggering of the data acquisition. No postacquisition resampling is needed, avoiding ghost images and reducing computation time. The akinetic laser has a typical RMS linearity of 0.0012% at 200,000 sweeps per second (<±1pm; ±0.2GHz) and a linearity span that is typically <±0.002% (<±2pm). Wavelength repeatability of 0.5 pm (standard deviation) and ±2.5pm span (peak amplitude) have been confirmed for the all-semiconductor laser. In air, a wavelength repeatability of 0.5 pm produces a phase error of 0.5 mrad at 0.2 mm (5 mrad at 2 mm). Coherence revival occurs if a laser is simultaneously oscillating at multiple longitudinal modes of the cavity. Because the akinetic swept laser has a single longitudinal mode, the issues with coherence revival in mechanically tuned lasers are largely avoided. Output power in the all-semiconductor akinetic laser is directly controlled on the chip and can be selected from software. Over the life of a laser, the center wavelength will only vary by 80pm based on life-test measurements taken on similar laser devices. The nonactive coherence length of the all-semiconductor laser is in the range of several tens of meters. This is substantially higher coherence than the 200mm of currently available all-semiconductor lasers. Main envisaged innovations are user-adjusted coherence length and a software platform allowing detailed control over the laser’s behavior. In lab experiments, all-semiconductor lasers have been demonstrated at 1 million sweeps per second. In the long run, it is believed that the akinetic technology can be pushed to roughly 2 million sweeps per second. With an akinetic laser, other power profiles may be tried by the user, under program control, for application-specific optimization without any other changes to the system. The same technology (with a modified substrate) will be employed for 1060 nm. The electronic drive circuitry is identical for 1310, 1550, and 1060 nm. It is also within the bounds of the technology to work with applications at 850 and >1640nm. The all-semiconductor technology will allow the production of lasers with wavelength coverage of 140nm and wider in the future. The wafer-scale laser manufacturing technology used for the all-semiconductor laser also grants cost-effectiveness to this product. Use of high-volume telecom supplies for the internal electronics may further reduce the costs for this type of laser.

Santec Corporation, Komaki, Japan

Santec is one of the first companies to launch a swept source especially optimized for OCT imaging: an external cavity laser (ECL) with a polygon mirror and a diffraction grating, resulting in high-performance sources with a desirable linear sweep behavior in k (frequency domain), but suffering from insufficient scaling toward higher speed due to the bulkiness of the polygon mirror, which, in combination with the high price, prevents widespread cost-effective use (information in this section is from www.santec.com). ECLs with a polygon mirror are available at 1300 nm (20kHz/>170nm/>20mW; 50kHz/140nm/15 to 30 mW; 1060 and 1310nm/60mm double pass coherence length/1 to 3kHz/6 to 8 mW; 0.15% linearity/1310nm/20kHz/8mm coherence length>20mW) and at 1050 nm (30 to 50kHz/70 to 120nm/>12mW/6mm double pass coherence length). These sources will remain research/academic light sources due to aforementioned reasons. Santec also offers MEMS-based swept sources—at least at 1310 nm so far: 100kHz/>20mm double pass coherence length (6dB)/105nm sweep range/>50W optical output power, which is also available as an OEM product. This 1310 nm MEMS-based swept source is very similar to the Axsun 1310 nm swept source and is, therefore, considered a proper back-up by some OCT companies. Main envisaged innovations include an MEMS-based swept source at 1050 nm.

Thorlabs Inc., Newton, New Jersey

These lasers are the first (in 2012) commercially available MEMS vertical-cavity surface-emitting laser (MEMS-VCSEL) swept sources at 1050 nm (100 nm sweep range at 10dB) and 1310 nm (150 nm at 10dB sweep range) (information in this section is from www.thorlabs.us). The current tuning range of 150 nm at 1310 nm is the largest reported for any MEMS-VCSEL and is comparable to the 160 nm reported for the FD mode-locked (FDML) laser. First demonstrations of coherence lengths >100mm and axial scan rates up to 1.2 MHz have been accomplished. Ultrahigh speed, high-resolution imaging (up to 580 kHz), high-speed, long-depth range imaging (100 kHz), and ultra long-range imaging (50 kHz) have been demonstrated. A further advantage of the MEMS-VCSEL is wavelength flexibility. Adaptation of the same technology to other wavelengths from 450 to 2300 nm appears to be feasible. The VCSEL is optically pumped at 980 nm (for 1310 nm) or 850 nm (for 1050 nm) through a top dielectric mirror, generating tunable 1310 nm emission, which emerges from the top mirror and is fiber-coupled and amplified by a low-noise semiconductor optical amplifier. Efforts to date have focused on optically, rather than electrically, pumped devices—this makes the source more complex and expensive. Though the ultimate low-cost device will be electrically pumped, optical pumping provides a number of performance advantages over electrical pumping, including larger tuning range and better spectral purity. However, MEMS-VCSEL, like other MEMS-based tunable lasers, can be linearized through drive waveform preshaping. Linearized drive waveforms at 200 kHz axial scan rates have also been demonstrated. The duty cycle is >90% with symmetric forward and backward sweeps. Unidirectional frequency sweep of this source results in a 400 kHz A-scan rate with a duty cycle of 50% at 1050 nm. The system phase stability, defined as the standard deviation of the phase differences between sequential A-scans, measured from a mirror in the patient interface at a signal-to-noise ratio of 57.5 dB (ratio of peak to mean noise) and a depth of 0.3 mm was 1.5 mrad, which approaches the theoretical limit. Main envisaged innovations include MEMS actuator designs to increase mechanical resonance frequency to support higher-frequency drive, development of electrical instead of optical pumping for VCSELs for lower cost and miniaturization (since it would eliminate the pump, wavelength division multiplexer coupler, and isolator), and higher bandwidth and optical power at 1050 nm with improved booster amplifier. A further increase of the tuning range to 200 nm may be possible by increasing the number of quantum wells in the gain region, using a wider bandwidth top suspended mirror, and by further increasing the free spectral range. In addition, it should also be possible to multiplex two or more VCSELs with offset bandwidths in order to obtain increased sweep ranges and improve axial resolutions.

Exalos AG, Schlieren, Switzerland (Exalos Inc., Pennsylvania)

These lasers are ECLs with a resonant MEMS-based one-dimensional (1-D) scanning mirror and a diffraction grating, resulting in fast tunable sources with demonstrated sweep frequencies up to 200 kHz (information in this section is from www.exalos.com). Due to the use of high-Q resonant MEMS scanners, those sources perform a quasi-sinusoidal sweep in k, which requires linearization and signal postprocessing. However, the sweep behavior is highly deterministic and provides ultrastable long-term phase stability. The laser architecture itself is similar to polygon mirrors, flexible in wavelength, and allows for realizing swept sources from the visible to the near-infrared (NIR). Due to the miniaturized filter architecture, this approach allows for compact, cost-effective swept sources. At the moment, the company is the only provider that offers swept sources between 400 and 1700 nm with sweep rates from 2 to 200 kHz in a miniaturized optical butterfly package that also allows the integration of optical reference (k-clock) interferometers or other optical reference filters for spectral calibration. 10-dB sweep ranges as wide as 80 nm at 840 nm, 120 nm at 1060 nm, 100 nm at 1220 nm, 150 nm at 1300 nm, and 200 nm at 1550 nm, respectively, have been achieved. Clean imaging performance results in no secondary coherence peaks as well as sharp and narrow point spread function peaks without side lobes throughout the imaging. Main envisaged innovations include next-generation ultrahigh-speed 1-D MEMS scanners with mechanical resonance frequencies in the range of 75 to 150 kHz, resulting in 150 to 300 kHz swept sources. Ultra-broadband semiconductor optical gain chips at 840, 1050, and 1310 nm enable ultrawide optical sweep ranges and hence unprecedented axial resolution.

Fourier Domain Mode-Locked Laser

So far, the main impact of FDML lasers has been the demonstration of OCT systems with dramatically higher imaging speed.21,22,7678 The first versions have pushed the speed from several 10 kHz line rate, which have been standard for the first FD OCT systems, to several 100 kHz, and later on, FDML lasers have helped to break the barrier of a 1 MHz line rate with swept sources. Besides the higher imaging speed, FDML lasers have been proven useful for many different applications, where good phase stability, long coherence, low laser noise, or similar conditions are required. Despite these many initial applications, only a few applied or clinical studies using FDML have been published. This is partly caused by the difficulty to build proper OCT systems that can handle the high imaging speed and the fast generated huge data sets by these multi-megahertz (MHz) OCT systems. It is interesting to see how the high-speed FDML results have triggered vibrant research efforts to realize non-FDML sources, which can achieve similar performance. It can be expected that the availability of more than one swept laser technology for MHz OCT will spur research on applications to find out where MHz OCT imaging speeds are required. FDML lasers can achieve ultrahigh sweep rates of up to 5.2 MHz by buffering or multiplexing the sweeps. FDML works optimally at 1.3 and 1.5 μm wavelengths where optical fiber dispersion and loss are negligible. However, dispersion can be compensated for using fiber Bragg gratings to improve performance at 1 and 1.3 μm wavelengths. The fact that the laser light is seeded from the last round-trip and that FDML is a real stationary laser-operating regime reduces the RIN of the laser. The good saturation of the laser gain medium enables very high output powers of 100 mW and more.

Necessary swept source technological specifications for future envisaged (commercial) OCT systems in the coming years would be at least 200 to 400 kHz (with preferred unidirectional sweep), with a 100 to 150 nm sweep range (ideally not at 10dB but as flattop in linear scale and with good optical flatness), optical output power >10mW, coherence length of 100 to 150 mm, duty cycle (>90%), high phase stability, linear-in-frequency sweep (picometer range; tenths of gigahertz), and low RIN noise (150dB/Hz). At the moment, the perfect swept source for (commercial) OCT in terms of optimum specs, size, price, robustness, and future potential does not exist.

The akinetic, all-semiconductor Insight swept source is extremely easy to use, very flexible, compact, suitable for a miniaturized OCT system (OCT on a chip), and has the potential to be very economical with unprecedented performance. However, it has only been partially explored from an OCT imaging point of view. The VCSEL MEMS-based swept source is no doubt the most developed long-coherence high-speed swept source at the moment. Despite its momentary impressive performance, the short micron scale length of the VCSEL cavity will probably be a risk of generating artifacts due to slight misalignments or long-term instabilities. Furthermore, electrical pumping would be preferred to reduce complexity, costs, and size. Exalos is currently the only provider for swept sources at all five wavelength regions, also including 850 nm. Most of the swept source technology companies entered the market only quite recently. Therefore, an absolutely fair judgment regarding lifetime, stability, and other specifications is challenging. In general, nonmechanical/akinetic swept sources should be a better choice due to the proneness to failure of the mechanical scanning mechanism. The NNT-AT KTN-based swept source is nonmechanical, but seems to be significantly harder to operate and does not promise flexibility and performance features like the akinetic all-semiconductor, akinetic Insight swept source.

During the last two decades, OCT has seen dramatic improvements in resolution, sensitivity, and speed. For in vivo imaging, resolution and speed are strongly linked, since motion blurring degrades not only resolution, but also sensitivity. The faster the acquisition, the shorter is the measurement time, which is, in particular, important for human in vivo imaging. The speed performance improvement is impressive: first implementations of OCT systems achieved a few depth scans per second. Nowadays, based on rapidly tuning SS technology, even several millions of such A-scans have been demonstrated. This allows, on one hand, dense sampling of large tissue patches, allowing for a comprehensive insight into tissue morphology, and, on the other hand, new functional imaging modalities profited from an enhanced flexibility. Nevertheless, even ultrafast systems ultimately strike the physical boundaries given by the detection process. Especially for in vivo imaging, the applied optical power is limited by laser safety regulations. Increasing, therefore, the speed, i.e., reducing the detection time, without being able to adjust the applied power, reduces the sensitivity, and degrades contrast and image quality. It is, therefore, questionable if systems much faster than 100 to 400 kHz will be used for clinical OCT applications.

A natural way out of this dilemma is to parallelize the detection. The theoretical advantage of full-field parallel OCT (FF OCT) regarding its intrinsic better sensitivity and potentially higher speed has been recognized already with early time domain OCT (TD OCT). Sensitivity in the shot noise limit scales proportional with the number of detected photons backscattered from the sample. It increases, therefore, with sensor recording time and with optical power. Flying-spot OCT covers each sampling point for a shorter time as the speed is increased. Parallel recording does not suffer from this limitation. In fact, if the time needed for a flying-spot system to sample a tomogram of, let us say, N lateral points is T, then each scan has a recording time of only T/N. In parallel OCT, in contrast, the full time T is available for recording at all parallel points. This compensates for the spread of power across the parallel detection channels. The advantage in sensitivity for a parallel system comes now from the possibility to apply more power while still complying with laser safety regulations, since a larger patch of the sample is illuminated. Although feasibility of parallelization of OCT for in vivo imaging has been demonstrated, there are some serious challenges that need to be resolved in the future to make it suitable for practical clinical applications. In the following, we will review developments in FF and line-field (LF) OCT and discuss current limitations and possible solutions for establishing parallel OCT and exploiting its potential advantages.

Full-Field Time Domain Optical Coherence Microscopy/Tomography

Conventional TD OCT uses a low-coherence light source to achieve micrometer axial resolution. The light is focused on the sample using a low numerical aperture (NA) microscope objective (MO) to keep the confocal gate long enough to cover the full imaging range in the sample. This avoids the need to move the sample in the Z-direction. Instead, the sample at a given lateral point is scanned in depth by modulating the optical path length of the reference arm at a high speed. By scanning the sample in the lateral direction, in vivo B-scans (X-Z cross-section) with high axial resolution can be obtained.79 But, the use of a low NA sacrifices the lateral resolution, which is typically on the order of 10 to 30 μm. Beaurepaire et al.80 demonstrated that by simultaneously irradiating the FF of view on the sample with a low-coherence light source and detecting the backscattered light using a high-NA MO and a two-dimensional (2-D) charge-coupled device (CCD) array in a Michelson interferometric setup, a 2-D en face image (X-Y cross-section) with high lateral resolution can be obtained. A lateral resolution of two was achieved using an MO of NA=0.25. The axial resolution was 8 μm, limited by the low-coherence light-emitting diode light source with a center wavelength at λ0=840nm and bandwidth Δλ=31nm (output power=20mW). As the confocal gating was narrow due to the MO with a high NA, the sample was scanned in depth to several hundred microns by moving in the sample in the Z-direction to create a three-dimensional (3-D) volume image. The magnitude of the interferometric signal from each pixel of the CCD array was extracted in parallel using a lock-in detection technique.80,81 A combination of linearly polarized illumination and modulation of detected interferometric data using a photo-elastic birefringence modulator and an analyzer (45 deg) were used to create phase-shifted data.80 A linear combination of four phase-shifted data (phase shifted by 0, 90, 180, and 270 deg) is used to produce a signal at a given pixel, proportional to Asin(2πδ/λ) and Acos(2πδ/λ), where λ is the center wavelength of the illumination, A is the amplitude of the coherent backscattered light, and δ is the optical path length difference between the light from the reference mirror and backscatters in the corresponding sample voxel.80 The CCD camera (Dalsa, CA-D1, pixels) offered a frame rate of 200 Hz, but the frame grabber (IC-PCI, Imaging Technology Inc.) limited the frame rate to 50 Hz. Sixty-four quadruplet images were acquired to produce an en face image [500×500μm2 field of view (FOV)] in 5 s with a reported sensitivity of 100dB (300 μW power on sample). Images of plant cells, such as onion cells, were shown up to the depth of 200 to 350 μm. Dubois et al.82 further improved the resolution of the system by modifying the system into a Michelson interferometer with a Linnik type configuration with MOs of the same NA in both the reference and the sample arms. The same light source, signal modulation, and detection system were used as in 80. A higher lateral resolution of 0.7 μm and axial resolution of 2.8 μm were reported using the MO with a high NA of 0.5. A depth penetration of only up to 150 μm was achieved. The lateral resolution degradation with depth was attributed to the increase in multiple scattering of light with depth in the sample. The measured sensitivity of the system was reported to be 82dB. Enface images of onion cells with fields of view of 160×160μm2 and 370×370μm2 were acquired in 0.5 and 1 s, respectively. However, the speed was not enough to image a moving sample, and thus, the imaging was restricted to ex vivo samples. Grieve et al.83 demonstrated 3-D ocular tissue imaging with cellular-level resolution using an ultrahigh-resolution FF TD OCT setup. The system was based on a Michelson interferometer with a Linnik type configuration. A tungsten halogen lamp was used as a light source with a center wavelength of λo=770nm and a bandwidth of Δλ=350nm. The low coherence of the light source provided a very high axial resolution of 0.7 μm. A high lateral resolution of 0.9 μm was achieved using an MO with NA=0.5. A piezoelectric transducer (PZT) in the reference arm was used to modulate the optical path length in the reference arm to produce the phase-shifted images. An en face image (500×500pixels) was acquired in 1 s. However, again due to the limited imaging speed, only ex vivo ocular tissue samples (cornea, lens, retina, choroid, and sclera) of rat, mouse, and pig were imaged. It was reported that a sample displacement of <1μm was necessary during the time scale of image acquisition.83 Oh et al. demonstrated better depth penetration in tissue by using a Xenon arc lamp as a light source and an InGaAs camera (SU320 MSW-RS170, 12 bit, 60 Hz) in an ultrahigh-resolution FF OCT setup.84 InGaAs cameras have better spectral response in the wavelength range of the Xenon lamp source (0.9 to 0.4 μm wavelength range) as compared to Si-cameras. Moreover, at longer wavelengths, the effect of multiple scattering is reduced. Depth penetration of 800μm was demonstrated by this setup in human thyroid tissue. The reported axial and lateral resolutions achieved were 1.9 and 2 μm, respectively.85 An en face image was recorded in 2 s with a sensitivity of 86 dB. Fig. 1 shows the TD FF OCT system based on linnik configuration used by Boccarra et al.