CROSS-REFERENCE TO PRIORITY APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 62/451,468, filed on January 27, 2017, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government funding under National Institute for Health Grant Numbers P30 EY07551; P30 EY003039; 2R01 EY022362. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present disclosure relates to imaging systems, and in particular to common- path phase-sensitive optical coherence tomography for enhanced phase stability and detection sensitivity.

BACKGROUND

[0004] Optical coherence tomography (OCT) is an interferometric imaging technique that allows for high-resolution, volumetric imaging of biological tissue. In conventional OCT, a low-coherent electromagnetic radiation (e.g., light) from a broadband source is divided into a reference path and into a sample path. The interference pattern as a result of the superposition of back-reflected electromagnetic radiation from the sample as well as the reference path contains information about the scattering amplitude as well as the location of the scattering sites in the sample to provide spatial image resolution in both lateral and axial dimensions.

[0005] With the development of OCT, the application optical coherence elastography (OCE) has gained more and more attention in biomechanics for its unique features including micron-scale resolution, real-time processing, and non-invasive imaging. OCE is comprised of a loading system to induce tissue mechanical waves and a high-resolution OCT imaging system to detect the resulting tissue deformation. If the tissue deformation induced during OCE imaging is on the order of microns, it may fall below the detection capabilities of conventional OCT imaging. By analyzing the complex component of the resulting

interferrograms, phase-sensitive OCT (PhS-OCT) imaging can provide much greater sample displacement sensitivity (nanometer-scale).

[0006] The improved displacement sensitivity of PhS-OCT relies upon the optical phase stability of the imaging system. However, dynamic turbulence existing between the sample and reference paths limits the optical phase stability, reduces deformation detection sensitivity, and degrades measurement precision. In particular, poor phase stability (micron- scale amplitude) can limit system performance and there are a number of potential sources of environmental, electromechanical, and optical turbulence that can reduce phase stability. For example, vibrational noise (e.g., seismic vibration, acoustic vibration, direct-force vibration, etc.) can exist between the sample and reference paths and is a major source of phase fluctuation. The vibration causes relative axial motion between the sample and reference paths, alters the interference path difference during data acquisition, and leads to an instable phase acquired from multiple lines or frames.

[0007] Post digital processing methods can be used to subtract the turbulence noise when the coupled signal and noise have either distinctive frequencies or amplitudes. However, digital processing methods cannot completely remove this noise and can induce signal measurement errors as well. Alternatively, the measurement beam can be split into a calibration mirror that is proximal to the sample path to provide a separate phase signal, which may then be subtracted from the phase modulation induced by the sample for phase calibration. This method essentially calibrates the system for major sources of phase instability, effectively isolating phase signals to the displacements of interest that might otherwise be dominated by non-signal vibrational fluctuations or other environmental and system noise. However, the different positions and mounting methods for the calibration mirror and the sample lead to different turbulence dynamics between them that can affect the phase calibration as well. Accordingly, the need exists for PhS-OCT systems with increased optical phase stability to provide greater detection sensitivity and displacement measurement repeatability.

BRIEF SUMMARY

[0008] In one embodiment, the invention relates to an optical imaging system including: a source for generating a coherent beam of electromagnetic radiation; a common optical path that shares a sample arm and a reference arm of the coherent beam of electromagnetic radiation; a reference plane defined by an optical surface for back-reflecting the reference arm of the coherent beam of electromagnetic radiation with a first optical signal; and a sample for back-reflecting the sample arm of the coherent beam of electromagnetic radiation with a second optical signal. The optical surface is located near or adjacent to the sample, and after back-reflecting, the reference arm and the sample arm are configured to interfere and create an interference signal based upon a phase difference between the first optical signal and the second optical signal and a time delay of the reference arm from the sample arm. The optical imaging system further includes a device for detecting and translating the inference signal into an interferogram.

[0009] In accordance with some aspects, the source is a low coherence, broad bandwidth electromagnetic radiation source. Optionally, the optical surface is within 1 mm - 12 mm from the sample.

[0010] In another embodiment, the invention relates to a common-path phase-sensitive optical coherence tomography system including: a low coherence, broad bandwidth electromagnetic radiation source for generating a coherent beam of electromagnetic radiation; a reference plane defined by an optical surface for back-reflecting a reference arm of the coherent beam of electromagnetic radiation with a first optical signal; and a sample for back- reflecting a sample arm of the coherent beam of electromagnetic radiation with a second optical signal. The optical surface is located near or adjacent to the sample. The common- path phase-sensitive optical coherence tomography system further includes a stimulator configured to excite transient surface waves in the sample. The transient surface waves create a phase shift between the first optical signal and the second optical signal, and after back- reflecting, the reference arm and the sample arm are configured to interfere and create an interference signal based upon the phase shift between the first optical signal and the second optical signal and a time delay of the reference arm from the sample arm. The common-path phase-sensitive optical coherence tomography system further includes a device for detecting and translating the interference signal.

[0011] In accordance with some aspects, the optical surface includes a coating configured to modulate an amount of the first optical signal back-reflecting from the reference plate as compared to the second optical signal back-reflecting from the sample.

[0012] In another embodiment, the invention relates to an optical system comprising: a common optical path that shares a sample arm and a reference arm of the coherent beam of electromagnetic radiation; a reference plane defined by an optical surface for back-reflecting the reference arm of the coherent beam of electromagnetic radiation with a first optical signal; and a sample for back-reflecting the sample arm of the coherent beam of

electromagnetic radiation with a second optical signal. The optical surface is located near or adjacent to the sample, and after back-reflecting, the reference arm and the sample arm are configured to interfere and create an interference signal based upon a phase difference between the first optical signal and the second optical signal and a time delay of the reference arm from the sample arm.

[0013] In accordance with some aspects, the optical system further includes a scan lens for producing a flat image plane with primary rays of different scan locations being parallel to an optical axis and perpendicular to the reference plate.

BRIEF DESCRIPTION OF THE DRAWINGS:

[0014] The present invention will be better understood in view of the following non- limiting figures, in which:

[0015] FIG. la shows a common-path PhS-OCT system in accordance with some aspects of the present invention;

[0016] FIG. lb shows a telecentric scan geometry of a common-path PhS-OCT system in accordance with some aspects of the present invention;

[0017] FIG. 2 shows a conventional PhS-OCT system;

[0018] FIG. 3(a-l) shows test results from a conventional PhS-OCE system;

[0019] FIG. 3(a-2) demonstrates the sinusoidal fitting for a test from conventional PhS- OCE measurements in accordance with some aspects of the present invention;

[0020] FIG. 3(b-l) shows test results from a common-path PhS-OCE system in accordance with some aspects of the present invention;

[0021] FIG. 3(b-2) demonstrates the sinusoidal fitting for a test from common-path PhS- OCE measurements in accordance with some aspects of the present invention;

[0022] FIG. 3(c) compares fitted frequencies in accordance with some aspects of the present invention;

[0023] FIGS. 3 (d-1 ) and 3(d-2) compare the fitted amplitudes for the measured phase noise in accordance with some aspects of the present invention;

[0024] FIG. 4(a) shows noise components (amplitude and frequency) from conventional PhS-OCE in accordance with some aspects of the present invention;

[0025] FIG. 4(b) shows noise components (amplitude and frequency) from common-path

PhS-OCE in accordance with some aspects of the present invention;

[0026] FIG. 5 shows phase instability in accordance with some aspects of the present invention;

[0027] FIG. 6(a) shows signal sensitivity fall off, measured from 0.33mm to 6.66mm at an interval of 0.33mm in accordance with some aspects of the present invention; [0028] FIG. 6(b) shows phase stability measurement (standard deviation of phase differences) at the corresponding depths (n=20) in accordance with some aspects of the present invention;

[0029] FIG. 7(a) shows signal sensitivity in the lateral scan locations from -5mm to 5mm in accordance with some aspects of the present invention;

[0030] FIG. 7(b) shows phase stability measurement (standard deviation of phase differences) at the corresponding scan locations (n=20)in accordance with some aspects of the present invention;

[0031] FIG. 8(a-l) shows the raw surface displacement data from conventional PhS-OCE in accordance with some aspects of the present invention;

[0032] FIG. 8(a-2) shows modified surface displacement data in accordance with some aspects of the present invention;

[0033] FIG. 8(b) shows the raw surface displacement data from common-path PhS-OCE in accordance with some aspects of the present invention;

[0034] FIG. 8(c-l) shows the primary deformation response in accordance with some aspects of the present invention;

[0035] FIG. 8(c-2) shows the coefficient of variation (CV) for the measured primary deformation in accordance with some aspects of the present invention;

[0036] FIG. 9(a) shows a raw interferogram having multiple fringe frequencies in accordance with some aspects of the present invention;

[0037] FIG. 9(b) shows a A-scan signals in the time window of to - tw in accordance with some aspects of the present invention; and

[0038] FIG. 9(c) shows A-scan signal at the time reference time of to in accordance with some aspects of the present invention.

DETAILED DESCRIPTION

I. Introduction

[0039] In various embodiments, the present invention is directed to an imaging system including a low coherence, broad bandwidth electromagnetic radiation source for generating a coherent beam of electromagnetic radiation that is split into two separate beams, a reference arm and sample arm. The sample arm focuses into the sample being observed, and the reference arm reflects back from a reference plate. These two beams then interfere and cause either amplification or negation of the signal based upon the phase and time delay of the backscatter electromagnetic radiation from the sample. The interference signal created from the amplification or negation is detected and then translated into an interferogram by an instrument. A problem associated with conventional phase-sensitive OCT (PhS-OCT) systems, however, is that the sample arm and the reference arm are physically isolated from one another and environmental turbulence (e.g., vibration, temperature change, air flow, etc.) exists between the sample and reference arms, which is a major source of dynamic background phase noise. The dynamic background phase noise limits the optical phase stability, reduces deformation detection sensitivity, and degrades measurement precision in the conventional PhS-OCT systems.

[0040] To address these problems, the present invention is directed to imaging systems that have a common-path PhS-OCT system whereby the interference signal is produced by combing returned electromagnetic radiation from the sample and a reference plate that is positioned near or adjacent to the sample. Positioning the reference plate near or adjacent to the sample limits the physical isolation of the sample and reference arms, and thus significantly reduces environmental turbulence between the sample and reference arms. The reduction in the environmental turbulence results in an increases in the optical phase stability and the phase detection capability of the system. For example, one illustrative embodiment of the present disclosure comprises: a source for generating a coherent beam of electromagnetic radiation; a common optical path that shares a sample arm and a reference arm of the coherent beam of electromagnetic radiation; a reference plane defined by an optical surface for back-reflecting the reference arm of the coherent beam of electromagnetic radiation with a first optical signal; and a sample for back-reflecting the sample arm of the coherent beam of electromagnetic radiation with a second optical signal. The optical surface is located near or adjacent to the sample, and after back-reflecting, the reference arm and the sample arm are configured to interfere and create an interference signal based upon a phase difference between the first optical signal and the second optical signal and a time delay of the reference arm from the sample arm. The optical imaging system further includes a device for detecting and translating the inference signal into an interferogram (e.g. Fourier transformation).

[0041] Advantageously, these approaches provide common-path PhS-OCT systems that effectively reduce the amplitude of background dynamic optical phase instability to a sub- nanometer level. In particular, common-path PhS-OCT systems described herein are capable of providing a more stable baseline for surface displacement quantification, a larger dynamic detection range, and higher detection sensitivity in displacement measurement than conventional PhS-OCT. II. Common-Path Phase-Sensitive Optical Coherence Tomography Systems

[0042] FIG. la shows a common-path PhS-OCT system 100 in accordance with some aspects of the present invention. In some embodiments, the common-path PhS-OCT system 100 includes a low coherence, broad bandwidth electromagnetic radiation source 105 for generating a coherent beam of electromagnetic radiation 110 where a sample arm 112 and reference arm 115 share a common optical path 1 17 with a reference plane 120 defined by an optical surface near or adjacent to the sample 122. The source 105 may be a laser diode capable of generating near-infrared light (e.g., within +/- 10 nm of 800 nm). In certain embodiments, the source 105 is a broad band super luminescence diode (100 nm) with a waveband of 795 nm - 895 nm and a central wavelength at λο=845 nm.

[0043] The sample arm 112 focuses into the sample 122 being observed and reflects back from the sample 122, and the reference arm 115 reflects back from the optical surface of a reference plate 125 within the reference plane 120. As discussed herein, the reference plane 120 is located near or adjacent to the sample 122 and the thickness of the reference plate 120 is optimized to differentiate and optically separate correct interferometric signals. As used herein, "near" or "adjacent to" mean within close approximation (i.e., within 1 mm - 12 mm, for example, 6 mm) of one another but not in contact with one another. The material and/or the surface coating of the reference plate 125 (e.g., an index of refraction) may be selected or modified to optimize the interferometric signals, their optical path length, and the ratio of transmitted and reflected light. In some embodiments, the reference plate 125 is an acrylic plate having a thickness of 2 mm - 8 mm (e.g., a 5 mm acrylic plate) and an optical path length of about 3 mm - 12 mm (e.g., 7.42 mm), which is longer than the OCT depth range of 2 mm - 10 mm (e.g., 6.76 mm), so that the lens-side optical surface of the reference plate 125 does not contribute to the spectral interferrograms. In other embodiments, the reference plate 125 is cover glass or a substrate having a millimeter-scale thickness that is less than or equal to 1 mm and both the lens-side optical surface and the sample-side optical surface contribute to the spectral interferrograms.

[0044] In various embodiments, the reference plane 120 and the sample 122 may share the common optical path 117 with a millimeter-scale axial distance in air (e.g., 0.3-4 mm) so that the electromagnetic radiation impacting the reference plane 120 and the sample 122 have the same polarization and dispersion properties and similar turbulence dynamics. By locating the reference plane 120 near or adjacent to the sample 122 and having the reference plane 120 and the sample 122 share the common optical path 117, it is possible to insure similar turbulence dynamics between the sample and reference paths, minimizing the relative motion between them. This configuration provides a more stable optical phase and more precise surface displacement measurements as well as a simpler and more compact configuration that can benefit further laboratory and clinical OCT studies.

[0045] In various embodiments, the common optical path 117 passes through a collimator 127 for producing a beam of parallel rays of electromagnetic radiation 130, an iris 132 for controlling a diameter of the beam of electromagnetic radiation 130, optional scanning optics 135 for scanning the beam of electromagnetic radiation 130 over the sample 122 in one or two dimensions x or x-y, a scan lens 137 for producing a flat image plane with the primary rays of different scan locations being parallel to the optical axis and perpendicular to the reference plate 125, the reference plane 120 including a sample-side optical surface of the reference plate 125 for generating the reference arm 115, and a sample plane 140 including the sample 122 for generating the sample arm 112. The electromagnetic radiation 130 or primary rays may pass through the reference plate 125 and some fraction of the rays may reflect at one or more of the surfaces (e.g., the lens-side optical surface of the reference plate 125 and/or the sample-side surface of the reference plate 125, which serves as the reference plane 140). The electromagnetic radiation 130 or primary rays may be focused in the sample plane 140 and may hit at any point on the sample 122 or pass through any position of the reference plate 125. The optional scanning optics 135 may be used to alter the position of the current beam path (primary rays) to be incident on the sample 122 at any particular location. In some embodiments, the sample 122 is not illuminated at all points simultaneously, but scanned as in a point-wise raster scanning imaging system. In certain embodiments to optimize signal intensity and surface reflectivity for common-path PhS-OCT, the collimator 127 is a parabolic, spherical, doublet, or other form of collimator to maximize useable waveband and axial resolution, and the scan lens 137 is a telecentric scanning objective to enhance sample and reference plate reflectivity (see, e.g., FIG. lb) by providing normal scan geometry over the full lateral scan dimension.

[0046] The two beams 112 and 115 then interfere and cause either amplification or negation of the signal based upon the phase and time delay of the backscatter electromagnetic radiation (i.e., the sample arm 112) from the sample 122. The interference signal is generated by combining the reference and sample signals (e.g., a first optical signal within the reference arm and a second optical signal within the sample arm), and then is recorded by an instrument or a detector 142 as an interferogram. In some embodiments, the detector 142 is a spectrometer for spectral domain optical coherence tomography (SD-OCT). In other embodiments, the detector 142 is a balanced photo-detector for swept-source optical coherence tomography (SS-OCT)). Phase shifting information within the reference and sample signals can be resolved as a function of time or location that shows a sub-pixel difference in the axial distance between the reference plane and sample In particular, a portion of the beam of electromagnetic radiation 130 is back-reflected as the reference arm 115 from a reflector 144 of the reference plate 125 and recombined at a beam recombining device 145 with another portion of the beam of electromagnetic radiation 130 back-reflected as the sample arm 1 12 from the sample 122. This recombined electromagnetic radiation 147 is detected by the detector 142. A digitized signal including the interferogram at the output port of the detector 142 may be transferred to a central processing unit (CPU) 150 for further processing to generate an image of the sample 122.

[0047] The phase shifting may be generated from mechanical stimulation of the sample, which causes dynamic sample deformation between the reference plate and the sample. The detection sensitivity for the phase shifting (e.g., image sensitivity or phase sensitivity) is a function of relative intensity of the electromagnetic radiation in the reference arm and sample arm. For example, if the reference arm and sample arm do not have enough intensity or are overly weighted as compared to one another, then there will be low contrast in phase difference and poor image sensitivity. However, if the intensity between the reference arm and the sample arm can be manipulated to have enough intensity and maintain similar sensitivity between the reference arm and the sample arm (e.g., the reference arm has 50% intensity and the sample arm also has 50% intensity), then there will be good contrast in phase difference and improved image sensitivity. In accordance with various aspects of the present invention, phase sensitive detection can be optimized by enhancing the signal to noise ratio using an optical coating on the reference plate to modulate an amount of signal back reflecting from the reference plate as compared to the sample. In some embodiments, the coating may be applied to the reference plate to selectively alter the ratio of reflectivity between the reference arm and the sample arm such that intensity between the reference arm and the sample arm is optimized to achieve maximal contrast. The coating may be an anti- reflective coating that is selective to a band of interest such as bandpass, low pass, high pass, filters in order to optimize frequencies of light selected to pass through the reference plate.

[0048] In various embodiments, the detector 142 includes a collimator 152 for producing a beam of parallel rays of electromagnetic radiation 154, a grating 155 that splits and diffracts the beam of electromagnetic radiation 152 into its spectral components 160 travelling in different directions as a form of structural coloration, a focusing lens 165 for focusing each of the beams 160, and a detection component 167 (e.g., a ID-line or a 2D-array detector) that translates the intensity of the electromagnetic radiation from each beam into an electrical signal. In certain embodiments to incorporate a reference plate, scanning offset, and sample depth and still have sufficient range and sensitivity to acquire high-contrast phase signals, the collimator 145 is a two plano-convex collimating lenses to reduce field curvature for grating dispersed wavelengths. The detector 142 may be optimized to provide longer scan depth (e.g., 6.67 mm) and sufficient imaging sensitivity to permit the use of low-power infra-red energy that is below the thermal biological safety limit for skin and ocular tissues.

[0049] Phase changes can be detected by the detector 142 as a sub-pixel difference in the axial distance between the sample arm 1 12 and the reference arm 1 15. These observed phase differences include any background phase noise (system instability) as well as any induced tissue surface displacements during measurements. Phase information can be resolved by tracing one point from the sample surface among the successive A-scan signals. Phase can be converted to distance using Equation (1) shown as:

Ax(t _j - t ₀ ) A

4π (i)

Where φ (tj-to) is the unwrapped phase change from the time point of tj to the referenced time point of to among the successive A-scan signals, Δχ (tj-to) is the corresponding axial distance change, λο is the center wavelength.

[0050] In certain embodiments in which the common-path PhS-OCT system 100 is used in the application of OCE to perform common-path PhS-OCE, the common-path PhS-OCT system 100 further includes a stimulator 170 (e.g., a micro-scale air-pulse stimulator) that excites transient surface waves (e.g., localized tissue excitation from a low-pressure and short-duration air stream) in the sample 122. The transient surface waves may be used to create phase shifting and make non-contact measurements of tissue elasticity within the sample 122. In some embodiments, the reference plate 125 is mounted between the scan lens 137 and the sample 122, and includes an aperture 175 to permit excitation of the sample through the reference plate 125. Accordingly, the common-path PhS-OCT system 100 includes the sample 122, the reference plate 125, and the stimulator 170 in a common alignment geometry with (i) the reference plate 125 in focus range of the scan lens 137, within depth range of the detector 142, and far enough from the sample 122 that the reference plate 125 will not cause harm or damage to sample 122 (e.g., flat or smooth), and (ii) the stimulator 170 mechanically isolated from the optical imaging elements such as the scan lens 137 and the reference plate 125 to minimize or prevent interference with the optics.

Ill Phase Stability

[0051] Without intending to limit the scope of the present invention, the common-path PhS-OCT system of the present invention may be better understood by referring to the following example(s).

[0052] Phase detection sensitivity of a PhS-OCT system relies on phase stability. This section focuses on the evaluation of the phase stability for conventional PhS-OCE (e.g., a conventional PhS-OCT with physically separated sample and reference arms as shown in FIG. 2 performing common-path PhS-OCE) and common-path PhS-OCE (e.g., a common- path PhS-OCT system 100 as described with respect to FIG. 1 performing common-path PhS- OCE).

Quantification of Phase Noise for Conventional and Common-Path PhS-OCT

[0053] In Example (1), a mirror served as the sample to evaluate phase stability for both conventional PhS-OCE and common-path PhS-OCE systems. Phase noise was quantified for each system under the same test conditions based on 9-total measurements. The temporal resolution was 15 and the analysis time window was 90 ms for each test.

[0054] FIG. 3(a-l) shows the background phase noise for conventional PhS-OCE measurements (Tests 1-9), plotted from top to bottom. Each test was shifted along the vertical axis by 40 radians (2.69 μπι) to reduce overlap and improve visualization. The dominant component of the phase variation measurement was a low-frequency periodic signal. This low-frequency feature had been previously identified as environmental turbulence (predominantly vibrations) between the sample and reference arms. This low frequency phase noise was dampened in amplitude when the optical table was floated (data not shown).

[0055] FIG. 3(b-l) shows the measured background noise for common-path PhS-OCE (Tests 1-9), plotted from top to bottom. Each measure was shifted along the vertical axis in an interval of 0.02 radians (1.3 nm) to reduce overlap. Similar low-frequency phase variations were measured in common-path PhS-OCE, but the amplitude was reduced by several orders of magnitude. [0056] To quantify the dominant low-frequency components for the phase fluctuations, a sinusoidal curve fitting method was used based on Equation (2) shown as:

y = y _c + A sm[27 (x - x _c ) (2)

Where /is the dominant frequency, A is the dominant amplitude; x represents time and .y represents phase or displacement, relative to the central coordinate of (x _c , y _c ).

[0057] FIG. 3(a-2) demonstrates the sinusoidal fitting for Test 1 from conventional PhS- OCE measurements. FIG. 3(b-2) demonstrates the sinusoidal fitting for Test 1 from common- path PhS-OCE measurements. The phase noise was fitted to sinusoidal curves with mean 95% confidence interval (CI) errors of ±0.0083 Hz and ±0.4233 Hz in frequency, and 0.0322 radians (2.2 nm) and 0.32 milliradians (0.02 nm) in amplitude for conventional PhS-OCE and common-path PhS-OCE measurements, respectively.

[0058] Fig. 3(c) compares the fitted frequencies for the phase noise acquired from each PhS-OCE system. The measured conventional PhS-OCE noise frequency was 20.68 - 21.62 Hz (95% CI: 21.05 ± 0.20 Hz). The measured common-path PhS-OCE noise frequency was 14.32 - 26.02 Hz (95% CI: 21.01 ± 3.03 Hz). Both OCE measurement techniques showed a ~ 21 Hz vibrational turbulence in the lab environment.

[0059] FIG. 3(d-l) and FIG. 3(d-2) compare the fitted amplitudes for the phase noise acquired from both conventional PhS-OCE and common-path PhS-OCE measurement techniques. The measured conventional PhS-OCE noise amplitude was 20.60 - 28.36 radians (95% CI: 23.77 ± 1.67 radians). This corresponds to 1.39 - 1.91 μιη (95% CI: 1.60 ± 0.1 1 μπι). The measured common-path PhS-OCE phase noise amplitude was 1.8 - 7.0 milliradians (95% CI: 3.5 ± 1.05 milliradians). This corresponds to 0.12 - 0.47 nm (95% CI: 0.24 ± 0.07 nm). As shown, the common-path technique reduces the amplitude of the dominant low- frequency noise component from tens of radians (micrometer scale) in conventional PhS- OCE to a milliradians (sub-nanometer scale) in common-path PhS-OCE.

[0060] A frequency analysis of the observed background phase noise was performed using a Fourier transform (FFT) method for conventional PhS-OCE and common-path PhS- OCE in FIG. 4(a) and FIG. 4(b), respectively. The frequency resolution of the FFT was 1 1.1 1 Hz. The FFT results show similar frequency components in the measured phase noise by each PhS-OCE technique: -22.22 Hz and -1.5 kHz. For the -22.22 Hz noise component, the amplitude was (mean±95%CI) 23.33 ± 1.58 radians (1.57 ± 0.1 1 μπι) in conventional PhS- OCE, and was 3.1 ± 0.91 milliradians (0.21 ± 0.06 nm) in common-path PhS-OCE. This was consistent with the sinusoidal fitting results. For the -1.5 kHz components, the amplitude was 64.65 ± 6.33 milliradians (4.35 ± 0.43 nm) in conventional PhS-OCE, and was 1.01 ± 0.16 milliradians (0.07 ± 0.01 nm) in common-path PhS-OCE. As shown, the common-path PhS- OCE technique effectively reduced the amplitude of all phase noise frequencies.

Phase Stability at Different Data Acquisition Speeds

[0061] As should be understood, faster camera speed (i.e., less exposure time) reduces the data acquisition time, excitation repetitions, and laser exposure at the expense of lower intensity-detection sensitivity.

[0062] In Example (2), to evaluate the phase stability at different A-scan acquisition speeds (camera speeds) of common-path PhS-OCE, the background phase noise was quantified based on the standard deviation value at the detector (e.g., a camera) speeds from 140 kHz to 16.7 kHz and in the time windows from 5 ms to 320 ms. The detector gain was set as zero for consistent evaluations. Each condition was tested five times. The detector (a spL4096-140kmESC camera, Basler) had two pixel lines. Dual-line acquisition mode had a maximum speed of 140 kHz while single-line acquisition mode had a maximum speed of 70 kHz. For higher speeds of 140 kHz, 100 kHz and 83.3 kHz, dual-line acquisition mode was selected. For slower speeds of 66.7 kHz, 33.3 kHz, 22.2 kHz and 16.7 kHz, single-line acquisition mode was selected.

[0063] The test results of phase instability for common-path PhS-OCE are shown in FIG. 5. It was noted that the phase was more stable when the camera ran at slower speeds (e.g., 16.7 - 100 kHz) rather than the highest speed (140 kHz). The standard deviation for phase variations at speeds of 16.7 - 100 kHz were near or below 5 milliradians (0.33 nm). The largest standard deviation value in phase was 23.33 milliradians (1.57nm) measured at the camera speed of 140 kHz and in the time window of 5 ms, which was also in the nanometer scale. Generally, the camera was run at a 66.7 kHz speed and in a single-line acquisition mode, which provided a good balance between speed, structure-detection sensitivity, phase stability, and phase-detection sensitivity. The phase standard deviation at 66.7 kHz speed with a time windows of 5 - 320 ms was (mean±95%CI) 3.23 ± 0.19 milliradians (0.22 ± 0.01 nm).

Phase Stability at Different Depths and Scanning Locations

[0064] As should be understood, a fundamental limitation on phase detection capability arises from signal-to-noise ratio (S R) of the intensity imaging, reported as [0065] In Example (3), the S R and the phase stability (as the standard deviation of phase differences) was compared and evaluated in various depths and scanning locations and camera speeds.

[0066] In SD-OCT, signal sensitivity tends to be weaker in deeper imaging regions. This depth-dependent loss in signal sensitivity is called "fall-off. The signal sensitivity at the depths of 0.33 - 6.66 mm was measured in using a common-path PhS-OCE system at an interval of 0.33 mm. The maximum measured signal sensitivity was 102.4 dB at the depth of 0.33 mm. The sensitivity fall-off relative to the depth of 0.33mm is shown in FIG. 6(a). The maximum fall-off was -36.0 dB at the depth of 6.66 mm and the -6 dB fall-off position was ~3 mm. Phase variations were measured 20 times at each depth and the standard deviation of each measurement is shown in FIG. 6(b). It was noted that even the maximum intensity sensitivity variation was -36.0 dB in this 6.33 mm depth range, the phase stability remains consistent with its mean and standard deviation values of 3.54 milliradians (0.24 nm) and 0.61 milliradians (0.04 nm).

[0067] Previous results were evaluated when the OCT beam was pointed at the same spots of the reference plane and the sample. As the beam was scanned [See, for example, FIG. 1(b)], the optical path lengths and point spread functions of different scan fields would also arise the differences in the detection signal sensitivity. For example, the signal sensitivity was measured in the lateral scan range of -5 mm to 5 mm and at the depth of 1.65mm, as shown in FIG. 7(a). The measured scan interval was 1mm. The maximum sensitivity drop-off was -13.3 dB at the scan location of +5 mm, and the -6 dB fall-off scan range was ~ ±3 mm. Phase variations were measured 20 times at each scan location and the standard deviation of each measurement was plotted in FIG. 7(b). The phase stability also remains consistent across the scan range with the mean and standard deviation values of 3.05 milliradians (0.21 nm) and 0.48 milliradians (0.03 nm) respectively.

IV. Surface Displacement Measurements

[0068] Without intending to limit the scope of the present invention, the common-path PhS-OCT system of the present invention may be better understood by referring to the following example(s).

Material

[0069] In Example (4), displacement measurement comparisons for both conventional PhS-OCE and common-path PhS-OCE systems were performed with 2% agar phantoms, which were prepared in 35 mm petri dishes. The tissue mimicking phantoms were prepared, sealed, and cooled in a refrigerator overnight before use. The weight of the solution was controlled before and after heating to insure consistency when preparing the phantoms (errors < ± 0.01%). Additionally, the weight of the phantoms was tracked during the experiment to account for any evaporation or dehydration (0.01% - 0.04%).

Surface Displacement

[0070] M-mode surface displacement detection (repeated A-scan acquisitions as a function of time at the same location) was performed for both conventional PhS-OCE and common-path PhS-OCE systems with a temporal resolution of 15 μβ. The tip of the micro air pulse stimulator (150 μιη in diameter) was kept - 0.5 mm from the surface of the sample to reduce any near-field influence. The stimulus air pressures were increased from 4 Pa to 32 Pa with a step of 4 Pa to generate mechanical responses of small to large displacements in the 2% agar phantom. Focused air-pulses for each air pressure were delivered in 5-time repetitions with a cycle time of 500 ms. Phantom surface responses were detected by PhS- OCT in the center of the phantom and as a lateral distance of - 0.3 mm from the excitation point. The air-pulse stimulus was triggered at 10 ms over a total measurement period of 70 ms. Surface displacement of the agar sample in response to this stimulus began at 11.7 ms and achieved maximum deformation at 13.7 ms. The initial deformation is referred to herein as the primary deformation response. This maximum negative displacement was followed by a relaxation recovery response as the sample returns to the initial position, which is complete by about 16.2 ms. This relaxation recovery period is best characterized by an exponential decay function. This relaxation response period is followed by a longer duration period (>70 ms) of high-frequency low-amplitude oscillations that gradually dampen. The full dynamics of this described deformation response is shown in FIG. 8(a-l), FIG. 8(a-2), and FIG. 8(b).

[0071] FIG. 8(a-l) shows the raw surface displacement data from conventional PhS- OCE, plotted in the sequence of air-pulse pressures from 4 Pa (top) to 32 Pa (bottom); each plot was shifted by 10 radians (0.67 μπι) to reduce overlap. The background noise was clearly observed and can be fitted as a sinusoidal curve [Equation (2)] or with other periodic functions that represent the dominant frequencies and amplitudes, such as a first-order Fourier curve based on Equation (3) shown as:

γ = γ _ε +Α _ι οο$(2φ) + Α ₂ η(2φ). (3)

[0072] Equation (3) was chosen for fitting the background phase noise, which was (mean ±95%CI) 20.61 ± 0.52 Hz. This is consistent with previous frequency measurements, e.g., 21.05 ± 0.20 Hz in FIG. 8(c). The fitted background low-frequency phase noise was subtracted from the raw data in FIG. 8(a-l) and plotted the new surface displacement data in FIG. 8(a-2). It was noted that the majority of the background phase noise had been removed, but there was still some low-amplitude residual disturbance that could affect measurement precision.

[0073] FIG.8(b) shows the raw surface displacement data from common-path PhS-OCE, plotted in the sequence of air-pulse pressures from 4 Pa (top) to 32 Pa (bottom). These data are plotted with each series shifted by 10 radians (0.67 μιη) to avoid overlap. The phase instability has been reduced to a sub-nanometer level in common-path PhS-OCE so that it is hard to distinguish in FIG. 8(b).

[0074] The primary deformation response to an applied force is related to the mechanical properties of the material tested. The magnitude of the maximum primary deformation response was measured relative to the absolute phase at 11.70 ms (onset of the sample deformation response) in the raw displacement data from conventional PhS-OCE [FIG. 8(a- 1)]. The amplitude of these peak values was calculated relative to the mean baseline phase measurements obtained in the 10 ms period prior to sample stimulation. For conventional PhS-OCE baseline phase was determined after subtracting the low-frequency background noise as described before (see, e.g., FIG. 8(a-2)). The raw displacement data from common- path PhS-OCE were measured directly as there was negligible low-frequency background noise by this measurement method (see, e.g., FIG. 8(b)).

[0075] FIG. 8(c-l) shows the primary deformation response from each method. The primary deformations were measured as ~ 3 - 60 radians (~ 0.20 - 4.03 μιη) corresponding to air-pulse pressures of 4-32 Pa. For each test under any air-pulse stimulus, the peak measurement distribution from common-path PhS-OCE was clearly observed as the smallest.

[0076] FIG. 8(c-2) shows the coefficient of variation (CV) for the measured primary deformation in each test, which was calculated as the standard deviation divided by the mean. To quantify and compare the surface displacement measurement precision in these three sets of data, a criterion of CV < 5% was introduced. Using the raw data from conventional PhS- OCE, all calculated CVs for the measured primary deformation responses were greater than the 5% threshold. For conventional PhS-OCE (low-frequency noise-subtracted) with moderate sample stimulation force (12 - 24 Pa), the average CV was 3.5% and produced moderate sample deformations (~ 13 - 42 radians [~ 0.87 - 2.82 μτη]). However, the CV was comparatively large (18.6%) for small surface deformations (- 3 - 8 radians [~ 0.2 - 0.54 μτη]) produced by small sample stimulation forces (4 - 8 Pa). Similarly, large deformation amplitudes (~ 50 - 60 radians [~ 3.36 - 4.03 μιη]) were produced by larger sample stimulation forces (28 - 32 Pa) and these were more variable (CV=11.0%). Surface displacements measured using common-path PhS-OCE were consistently small; and all CVs were less than 5% with an average value of 1.89%.

[0077] Accordingly, by improving phase stability using a common-path technique, the measurement precision for the primary deformation response was improved as shown in FIG. 8(c-l) and FIG. 8(c-2). In particular, FIG. 8(c-l) and FIG. 8(c-2) show that common-path OCE provides a larger dynamic detection range with higher detection sensitivity in surface displacement measurement than conventional PhS-OCE. Moreover, it was possible to see additional oscillation details persisting for longer periods. For example, the oscillation can be observed until the end of the time window (70 ms) with high sensitivity in common-path PhS-OCE (see, e.g., FIG. 8(b)), but was undetectable after -35 ms in conventional PhS-OCE (see, e.g., FIG. 8(a-l) and FIG. 8(a-2)). Without being bound by theory, the detection of additional oscillation details may be beneficial for quantifying other biomechanical properties of tissue, such as damping behavior, natural frequency, etc.

V. Impact of Reference Plate Thickness

[0078] Without intending to limit the scope of the present invention, the common-path PhS-OCT system of the present invention may be better understood by referring to the following example(s).

[0079] In Examples (1), (2), (3) and (4), a 5 -mm thick acrylic plate was used as the reference plate. The lens-side optical surface of the acrylic plate did not contribute to the spectral interferrograms and only the sample-side surface served as the reference plane.

However, in certain embodiments, a thin plate (e.g., a cover glass or substrate less than or equal to 1 mm) can be used as the reference plate. In the instance of a thin reference plate, both the lens-side optical surface and the sample-side optical surface of the thin reference plate may contribute to the spectral interferrograms. Consequently, the relevant signal for the sample surface needs to be identified from among the multiple interference signals in the A- scan profile for phase stability evaluation or surface displacement quantification. The relevant signal for the sample surface can be identified by comparing the interference path lengths and the imaging depths.

[0080] For example, a round-trip optical path length (OPL) for any returned beam that is produced by multiple reflections among the two surfaces of reference plate and the sample can be expressed as Equation (4) shown as: OPL , j) = OPL _L _ _side + i x (2T _p n _p ) + y x [2d + 2Ax( _tj - f ₀ )], (4)

Where OPLL-side is the round-trip OPL for the beam reflected by the lens-side surface of the reference plate. T _p and n _p are the thickness and refractive index for the cover glass, i represents the times of reflections inside the reference plate (i = 0, 1, ...), and j represents the times of reflections between the bottom surface of the plate and the front surface of the sample (j = 0, 1, ...).

[0081] If the OPLs for two returned beams are OPL(im, jm) and OPL(in, jn) respectively, the optical path difference (OPD) between them can be expressed as Equation (5) shown as:

PD _{m n} = (i _m - /J x (2T _p n _p ) + ( _Jm - j _n ) x [2d + 2Ax( _tj - t ₀ )]. (5)

[0082] Ideally, once OPDm,n/2 is within the OCT depth range (±Zmx), the interference between these two beams is detectable. If pairs of return beams have the same OPD, the interferences produced by these pairs have the same fringe frequency in interferrograms and the intensity peaks are combined at the same depth in A-scan profile. By comparing

OPDm,n/2 with Zmx, the relevant signal can be identified from among multiple reflections.

[0083] FIG. 9 demonstrates Example (5) where a thin cover glass (16004-406, VWR) was used as the reference plate. T _p = 1mm, n _p ~ 1.5163 at the wavelength of 845 nm. T _p n _p < Z _mx (-6.76 mm). The raw interferrograms contained multiple fringe frequencies (see, e.g., FIG.9(a)) and the A-scan signals had three peaks located at different depths (see, e.g., FIG. 9(b) and FIG. 9(c)). When the distance (d) between the reference plate and sample was adjusted, the first peak remained at the 462 ^nd pixel (1.525mm), the second and the third peaks were shifted correspondingly while maintaining their constant pixel difference of 462±1 pixels (1.525±0.03mm). In FIG. 9(b) and FIG. 9(c), the second peak was located at the 810 ^th pixel (d= 2.673mm), and the third peak was located at the 1272 ^nd pixel (4.198mm). Based on their depths, these three intensity peaks can be identified accordingly. The first peak was produced by both surfaces of the cover glass; the second peak was produced by the sample- side surface of the cover glass and the sample; and the third peak was produced by the lens- side surface of the cover glass and the sample.

[0084] While the invention has been described in detail, modifications within the spirit and scope of the invention will be readily apparent to the skilled artisan. It should be understood that aspects of the invention and portions of various embodiments and various features recited above and/or in the appended claims may be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by the skilled artisan. Furthermore, the skilled artisan will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention.