Self-interference imaging methods in polarization-dependent optical coherence microscopy

Abstract

Self-interference approaches for optical coherence microscopy and optical coherence tomography. Embodiments include a light source, a sample arm, a reference arm (optional for self-interference), and a processing system. The light source provides source light partially polarized about a first polarization axis. The sample arm receives a scanned portion of the source light and the sample to be imaged, and comprises a sample arm quarter wave plate having a fast axis at an angle between 0° and 90°, and optionally 45°, with respect to the first polarization axis. The reference arm receives a portion of the source light, and comprises a reference arm quarter wave plate having a fast axis at an angle between± 0° - 45° with respect to the fast axis of the sample arm quarter wave plate. The processing system processes electrical signals corresponding to interference of light received from the reference arm and sample arm, as well as self-interference of light received from different layers of the sample.

Description

SELF-INTERFERENCE IMAGING METHODS IN POLARIZATION-DEPENDENT OPTICAL COHERENCE MICROSCOPY FIELD

[0001] This disclosure relates generally to imaging devices and methods. Certain embodiments relate to improvements to optical coherence microscopy and optical coherence tomography. Embodiments include self-interference approaches BACKGROUND

[0002] Although transparent, the cornea includes well-organized microstructures that contribute to its homeostasis and function, and its shape provides two-thirds of the optical power of the eye. This outermost element of the visual system is at risk of infections and other environmental factors. It is also vulnerable to a multitude of pathological conditions, including keratoconus, dry eye, and Fuchs endothelial corneal dystrophy (FECD), which can compromise the cornea’s integrity and function, leading to vision loss or even blindness. Keratoconus is a multifactorial disease that causes the cornea to locally thin and bulge into a cone shape, resulting in asymmetric astigmatism and impaired visual acuity. Although the etiology of keratoconus remains unknown, studies have reported the damage of corneal nerves associated with the rupture of the Bowman’s layer within the keratoconic area, leading to a direct interaction between epithelial cells and keratocytes. Nerve injury and altered sensation may therefore be involved in the progression of the disease. FECD on the other hand is an hereditary disorder that affects the corneal endothelium causing a progressive loss of endothelial cells and in most cases the excrescences of the Descemet’s membrane called guttae. This condition may progressively cause the entire cornea to swell leading to corneal edema, altered epithelial cells, and corneal nerve. Changes in keratocytes’ density and reflectivity have also been reported in corneas with FECD. In addition to these diseases, contact lens wear can affect the cornea integrity in several ways, including the thickness and morphology of the corneal epithelium, the integrity of meibomian gland, as well as the sensitivity of corneal nerves, especially in people with pre- existing corneal conditions or altered ocular surfaces such as dry eye.

[0003] Non-invasive imaging technologies that can identify biomarkers of the early stage of corneal infection and disease with cellular-level resolution may offer the opportunity to advance our understanding of disease and enable early intervention and monitoring. Advancements in in vivo confocal microscopy (IVCM) and optical coherence tomography IU-2023-024-02-WO(OCT) have enhanced the ability to detect and describe cellular characteristics of a multitude of corneal pathologies.

[0004] IVCM can produce high-resolution images of biological tissues and has developed into a vital clinical tool in several disciplines, including ophthalmology. The advantages of IVCM include the capacity to image all layers of the cornea with cellular-level detail. IVCM, however, comes with certain drawbacks including the low acquisition speed and the need for ocular lubrication to optically couple the microscopic objective lens with the cornea. This contact with the ocular surface not only causes discomfort for patients but also potentially disrupts the natural organization of corneal structures and especially corneal nerves through the activation of mechanotransduction pathways. Also, the lack of a real-time cross-sectional view of the cornea during image acquisition makes it difficult to align the microscopic objective lens with the cornea and identify the shallow focusing depth location of the beam in the tissue. Moreover, a highly trained professional operator is often necessary.

[0005] OCT on the other hand can produce non-contact three-dimensional (3D) images of tissues with micrometer-scale axial resolution. Advantages of OCT over IVCM may include higher acquisition speed, non-invasiveness, real-time display of cross-sectional images of the cornea, the ability to capture images of all layers of the cornea in a single shot as well as provide an accurate evaluation of the corneal thickness and curvature. However, the non-contact benefit of OCT may come at the expense of motion artifacts that may be addressed through subject stability and increased frame rate. Moreover, OCT usually operates with a low numerical aperture (N.A.) objective lens, yielding images with poor cellular details compared to IVCM.

[0006] Optical coherence microscopy (OCM) may combine the high axial resolution of OCT and the high lateral resolution of IVCM through the use of high N.A. objective lenses in OCT and may be a viable technique for imaging microstructures of the human cornea. Full-field OCM (FF-OCM) operates in a similar manner as IVCM by illuminating the cornea with a collimated beam of light. FF-OCM uses the coherence property of light to section different depths of the cornea by moving the reference mirror of the interferometer instead of the sample / objective lens as is the case in IVCM. FF-OCM may also share drawbacks with IVCM including the lack of real-time visualization of cross-sectional images of the cornea, which can be mitigated by integrating FF-OCM with spectral-domain OCT. Spectral-domain OCM (SD- OCM) may overcome some of the drawbacks of IVCM / FF-OCM and OCT by enabling real-timecross-sectional images of the cornea with a cellular-level resolution, thus offering a fast thorough assessment of corneal microstructures at different depths

[0007] Optical imaging techniques such as optical coherence tomography (OCT) enable the evaluation of 3D structural information inside samples. An advantage of OCT is its capability of providing independent control over axial and lateral resolutions. The axial resolution may depend on the source parameters such as for example central wavelength and spectral bandwidth, whereas the lateral resolution may be controlled by the numerical aperture (N.A.) of the focusing objective. OCT systems often operate with low N.A. objectives in order to leverage the full imaging depth enabled by the combination of source and detector. Such systems may limit the lateral resolution, e.g., to tens of micrometers. To enhance the lateral resolution, optical coherence microscopy (OCM), a variant of OCT operating with high N.A. objectives, may yield enhanced lateral resolution, e.g., on the order of 1-3 µm.

[0008] OCM can, for example, be used to image cellular features of tissue such as that of the cornea. However, although visualization of certain structures of the cornea may be possible with OCM, visualization of other structures such as superficial squamous epithelial cells, wing and basal epithelial cells may be compromised by reason of the relatively low backscattered light from these cells compared to a relatively high specular reflection / haze from the surface of the cornea, resulting in a poor imaging contrast of epithelial cells.

[0009] There remains a need for enhanced imaging devices and methods. For example, methodologies that provide enhanced imaging of corneal tissue, such as enhanced resolutions and depths, would be desirable. Especially desirable would be methodologies that reveal the visualization of the superficial squamous epithelial cells of the cornea, including the boundaries and the underlying wing and basal epithelial cells. SUMMARY

[0010] Disclosed embodiments include enhanced imaging methods, devices and systems. Embodiments include enhanced OCT and OCM imaging methodologies. For example, as a new approach of OCM, by polarization-dependent optical coherence microscopy (POCM), the polarization property of light may be leveraged to enable the visualization of all layers of samples such as the human cornea in vivo.

[0011] Embodiments of the disclosed imaging methodologies may be referred to as polarization-dependent optical coherence microscopy (POCM). The polarization property oflight is effectively used as a contrast enhancer in embodiments to mitigate the contribution of the specular reflection from an imaged sample. For example, when used to image corneal tissues, embodiments of the methodologies enhance the corneal surface in FD (Fourier domain)-OCT image formation and enable the high contrast imaging of wing and basal corneal epithelial cells. The polarization property of light may also be used in polarization-sensitive optical coherence tomography (PS-OCT) and cross-polarization OCT (CP-OCT) to measure the birefringence property of tissues as well as structural changes due to pathological conditions, in embodiments.

[0012] Unlike conventional or prior art PS-OCT and CP-OCT, in which certain polarization properties of light have been used to evaluate the birefringence of the sample, the approaches disclosed herein, including POCM, use the polarization of light to enhance the contrast of cellular features, and specifically in the context of samples with highly reflective surfaces such as the corneal surface.

[0013] In some embodiments, a partially polarized light source (e.g., without a linear polarizer) was used in the interferometer. A quarter-wave plate (QWP) with fast axis angled atగସ or 45° of the horizontal axis (e.g., when the polarization axis is horizontal; could be any other axis corresponding to the angular distribution of polarization of the source) is integrated into the sample arm to turn the horizontal and non-horizontal linear polarizations into circular and elliptical polarizations, respectively. This step minimized the reflected light from the surface of the tissue while enabling light to penetrate in deep layers of the tissue. After a round trip through the sample (and e.g., assuming no change in polarization was induced by the sample), the QWP will act as a half wave plate, rotating all incident polarization states symmetrically around the fast axis of the QWP. For example, the horizontal linear polarization will become vertical after the round trip through the sample. Another QWP may be integrated into the reference arm with its fast axis adjusted between 0 −గସ or 0 − 45° relative to the fast axis of the QWP in the sample arm (e.g., relative to theaxis when the partial polarization is about the horizontal axis) to evaluate the change in image contrast. Evaluation of prototypes has demonstrated advantages by the use of the QWP in the reference arm, including reduction of specular reflections and enhanced imaging. However, in some embodiments, certain advantages may be achieved without the use of the QWP in the reference arm.

[0014] Examples include a method for imaging a sample. Embodiments of the example comprise providing source light that is partially polarized about a first polarization axis; splittingthe source light into a reference beam and a sample beam; scanning an incident portion of the sample beam and directing the scanned incident portion of the sample beam to the sample about a sample path, and causing a reflected portion of the sample beam to be reflected from the sample, including rotating polarization of the incident portion and the reflected portion of the sample beam with respect to the first polarization axis by a sample path rotational amount to reduce reflections from surface portions of the sample that interfere with imaging of subsurface portions of the sample, and wherein the sample path rotational amount is 0° - 90°, and optionally 45°; directing an incident portion of the reference beam to a reflector about a reference path and causing a reflected portion of the reference beam to be reflected from the reflector, including rotating polarization of the incident portion and the reflected portion of the reference beam with respect to the first polarization axis by a reference path rotational amount with respect to the sample path rotational amount, and wherein the reference path rotational amount is ± 0° - 45° with respect to the sample path rotational amount; combining the reflected portion of the reference beam and the reflected portion of the sample beam to produce a combined beam; and detecting and processing the combined beam to generate information representative of an image of the sample over axial and lateral portions of the sample.

[0015] In some embodiments of the method, rotating polarization of the incident portion and the reflected portion of the sample beam includes rotating polarization by an optical component, optionally a quarter wave plate.

[0016] In any or all embodiments of the method, rotating polarization of the incident portion and the reflected portion of the reference beam includes rotating polarization by an optical component, optionally a quarter wave plate.

[0017] In any or all embodiments of the method, the sample path rotational amount is 45°.

[0018] In any or all embodiments of the method, the reference path rotational amount is 45° with respect to the sample path rotational amount (e.g., + 45° or - 45° with respect to the sample path rotational amount).

[0019] In any or all embodiments of the method, the source light is polarized by an amount less than 75 %.

[0020] In any or all embodiments of the method, the source light is polarized by an amount less than 50 %.

[0021] In any or all embodiments of the method, the method includes optical coherence microscopy.

[0022] In any or all embodiments of the method, the method includes optical coherence tomography. For example, embodiments of the method include one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

[0023] In any or all embodiments of the method, detecting and processing the combined beam includes generating an image of the sample over axial and lateral portions of the sample via self-interference from the reflected portion of the sample beam.

[0024] In any or all embodiments of the method, detecting and processing the combined beam includes generating an image of the sample over axial and lateral portions of the sample via interference between the reflected portions of the sample beam and the reference beam.

[0025] A second example includes a method for imaging a sample. Embodiments comprise: providing source light that is partially polarized about a first polarization axis, including a sample beam; scanning an incident portion of the sample beam and directing the scanned incident portion of the sample beam to the sample about a sample path, and causing a reflected portion of the sample beam to be reflected from the sample, including rotating polarization of the incident portion and the reflected portion of the sample beam with respect to the first polarization axis by a sample path rotational amount to reduce reflections from surface portions of the sample that interfere with imaging of subsurface portions of the sample, and wherein the sample path rotational amount is 0° - 90°, and optionally 45°; and detecting and processing the reflected portion of the sample beam via self-interference to generate information representative of an image of the sample over axial and lateral portions of the sample.

[0026] In any or all embodiments of the second example, rotating polarization of the incident portion and the reflected portion of the sample beam includes rotating polarization by an optical component, optionally a quarter wave plate.

[0027] In any or all embodiments of the second example, the sample path rotational amount is 45°.

[0028] In any or all embodiments of the second example, the source light is polarized by an amount less than 75 %.

[0029] In any or all embodiments of the second example, the source light is polarized by an amount less than 50 %.

[0030] In any or all embodiments of the second example, the method includes optical coherence microscopy.

[0031] In any or all embodiments of the second example, the method includes optical coherence tomography. For example, the method may include one or more of frequency domain optical coherence tomography or time domain optical coherence tomography. A third example includes a system for imaging a sample. Embodiments of the example comprise an optical system and a processing system. The optical system includes: a light source providing source light partially polarized (e.g., about a first polarization axis, which may be a horizontal axis in embodiments); a sample arm configured to receive a scanned portion of the source light and the sample to be imaged, the sample arm comprising a sample arm quarter wave plate having a fast axis oriented at an angle between 0° and 90°, and optionally 45°, with respect to the first polarization axis of the source light; a reference arm configured to receive a portion of the source light, the reference arm comprising a reference arm quarter wave plate having a fast axis oriented at an angle between ± 0° - 45° with respect to the fast axis of the sample arm quarter wave plate. The processing system processes the electrical signals corresponding to light received from the reference arm and the sample arm of the optical system, to produce information representative of a three-dimensional image, including depth and transverse information, of the sample.

[0032] In embodiments of the system, the sample arm quarter wave plate has a fast axis oriented at an angle of 45° with respect to the polarization axis of the source.

[0033] In any or all embodiments of the system, the reference arm quarter wave plate has a fast axis oriented at an angle of + 45° or - 45° with respect to the fast axis of the sample arm quarter wave plate.

[0034] In any or all embodiments of the system, the light source provides source light polarized by an amount less than 75%.

[0035] In any or all embodiments of the system, the light source provides source light polarized by an amount less than 50%.

[0036] In any or all embodiments of the system, the system is configured to provide optical coherence microscopy.

[0037] In any or all embodiments of the system, the system is configured to provide optical coherence tomography. For example, embodiments of the system may be configured toprovide one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

[0038] In any or all embodiments of the system, the self-interference between a surface of the imaged object and underlying structures (e.g., a corneal surface and underlying layers) can be used to produce high-contrast images (e.g., epithelial cells and the sub-basal nerve plexus in the case of a cornea).

[0039] In any or all embodiments of the system, the processing system produces the information representative of the three-dimensional image of the sample via self-interference from the light received from the sample beam.

[0040] In any or all embodiments of the system, the processing system produces the information representative of the three-dimensional image of the sample via interference from the light received from the reference arm and the sample arm.

[0041] A fourth example includes a system for imaging a sample. Embodiments comprise: an optical system, including: a light source providing source light partially polarized about a first polarization axis; a sample arm configured to receive a scanned portion of the source light and the sample to be imaged, the sample arm comprising a sample arm quarter wave plate having a fast axis oriented at an angle between 0° and 90°, and optionally 45°, with respect to the first polarization axis of the source light; and a processing system for processing electrical signals corresponding to light received from the sample arm of the optical system via self- interference, to produce information representative of a three-dimensional image, including depth and transverse information, of the sample.

[0042] In any or all embodiments of the system of the fourth example, the sample arm quarter wave plate has a fast axis oriented at an angle of 45° with respect to the first polarization axis.

[0043] In any or all embodiments of the system of the fourth example, the reference arm quarter wave plate has a fast axis oriented at an angle of + 45° or - 45° with respect to the fast axis of the sample arm quarter wave plate.

[0044] In any or all embodiments of the system of the fourth example, the light source provides source light polarized by an amount less than 75%.

[0045] In any or all embodiments of the system of the fourth example, the light source provides source light polarized by an amount less than 50%.

[0046] In any or all embodiments of the system of the fourth example, the system of any of claims 30-34, wherein the system is configured to provide optical coherence microscopy.

[0047] In any or all embodiments of the system of the fourth example, the system is configured to provide optical coherence tomography. For example, the system may be configured to provide one or more of frequency domain optical coherence tomography or time domain optical coherence tomography. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG.1 is a diagrammatic illustration of a polarization-dependent optical coherence microscopy (POCM) system in accordance with embodiments.

[0049] FIG.2 is an illustration of interference signals and corresponding depth profiles of three configurations of embodiments, while focusing on the front surface of a thin glass sample.

[0050] FIG. 3 is an illustration of images produced by different configurations of an imaging system in accordance with embodiments

[0051] FIG. 4 is an illustration of enface images of different layers of porcine cornea produced by imaging systems in accordance with embodiments.

[0052] FIG.5 is a diagrammatic illustration of a POCM system for in vivo corneal imaging, including a polarization controller (PC), fiber collimator (FC), quarter wave plate (QWP), dispersion compensation elements (DCE), diffractive grating (DG), mirror (M), achromatic doublet (ACD), microscope objective (Obj), beam splitter (BS), fixation target (FT) and imaging lens (IL), in accordance with embodiments.

[0053] FIG.6 is an illustration of a subject stabilization system in accordance with embodiments.

[0054] FIGs.7A-7D are exemplary POCM Enface images of a resolution chart over a field of view of 0.5 x 0.5 mm2with the corresponding intensity profiles over the 6th elements (horizontal and vertical orientations) of the 7th group in four configurations.

[0055] FIGs.8a1, 8a2, 8b1, 8b2, 8c1, 8c2, 8d1 and 8d2 illustrate exemplary POCM images of the corneas of two healthy subjects, in accordance with embodiments.

[0056] FIGs.9a1, 9b1, 9c1, 9d1, 9e1, 9a2, 9b2, 9c2, 9d2 and 9e2 illustrate exemplary POCM images of the anterior cornea of the two subjects from the self-interference region in FIG. 8, in accordance with embodiments.

[0057] FIG.10 is an illustration of interference signals from embodiments of the system in three different arrangements of the reference arm quarter wave plate, with the fast axis of the quarter wave plate in the sample arm set at 45° from horizontal. DETAILED DESCRIPTION

[0058] FIG.1 is a diagrammatic illustration of a polarization-dependent optical coherence tomography (POCT) imaging system 10 in accordance with embodiments. A partially polarized broadband light source 12, such as for example an M-T-850-HP-I, from Superlum, Ireland, produces a partially polarized source light beam. In embodiments, the source beam produced by the light source 12 has a central wavelength of 850 nm and a spectral bandwidth of 165 nm (corresponding to a theoretical axial resolution of ~ 2 µm). Other embodiments have other light sources that produce light beams having other characteristics suitable for the imaging methodology described herein. The source beam from light source 12 is transmitted to an interferometer 14, e.g., through a 1x2 fiber coupler 16 in the illustrated embodiments, via one or more optical waveguides. The common-path approach of the embodiments shown in FIG.1 may provide enhanced control over the polarization in a free space configuration, thus mitigating the need for a broadband polarization-maintained fiber at 850 nm, as well as the instability of the system due to the optical path length shift between the two arms (reference arm 18 and sample arm 20) of the interferometer 14.

[0059] The illustrated embodiments include a polarization controller 24 in the common- path fiber waveguide 26 to adjust the polarization state of the incoming light so that its dominant polarization is horizontal (e.g. a first polarization axis). A beam splitter 28 is used to split the light into the reference and sample arms 18, 20. In embodiments, the beam splitter 28 is 70:30 (T:R) non-polarizing beam splitter, and a larger percentage portion of the beam is directed to the sample arm 20. Dispersion between the two arms 18, 20 may be compensated using a combination of optical dispersion compensation elements 30 (shown for example as a DCE (e.g., LSM54DC1, Thorlabs, Newton, NJ), a diffraction grating DG (e.g., GR25-0608, Thorlabs, Newton, NJ), an achromatic doublet ACD1 (e.g., AC254-030-B, Thorlabs, Newton, NJ), and a flat mirror M (e.g., PFSQ10-03-P01, Thorlabs, Newton, NJ). Alternatively or additionally, other dispersion compensation methods, including for example numerical approaches, may be used in embodiments. In the illustrated embodiments, the reference arm 18 includes a quarter wave plate (QWP) 19, and the sample arm 20 includes a quarter wave (QWP) plate 21.

[0060] The degree of polarization provided by the light source 12 may differ in different embodiments of the imaging system 10. For example, the degree of polarization may be less than 75 %, or less than 50 %. Other embodiments include other amounts of polarizations, such as for example less than 100 %. Additionally or alternatively, the partial polarization of the light source can be provided by optical components separate from the light source 12.

[0061] The portion of the source beam directed into the sample arm 20 by the beam splitter 28 (e.g., an incident portion of a sample beam) is scanned by a scanner 42. In embodiments, scanner 42 is a dual-axis galvanometer-type scanner that scans the incident portion of the sample beam in x and y directions. The scanned incident portion of the sample beam is directed to the sample 44 in the sample arm 20 about a sample path. The portion of the sample beam reflected from the sample 44 (e.g., a reflected portion of the sample beam) is directed back to the beam splitter 28 about the sample path. The illustrated embodiment of sample arm 20 includes other optical components, such as for example, achromatic doublets ACD1 and ACD2, and a focusing objective (e.g., 10X obj.).

[0062] The QWP 21 in the sample arm 20 is positioned or otherwise configured to rotate polarization of the incident and reflected portions of the sample beam with respect to or about the dominant polarization axis of the source beam produced by the light source 12. In embodiments, the fast axis of the QWP 21 is oriented at an angle with respect to the polarization axis of the source beam by an amount (e.g., a sample path or sample arm amount) sufficient to materially and preferably substantially reduce reflections from surface portions of the sample 44. In embodiments, the QWP 21 is configured to rotate the incident and reflected portions of the sample beam by an angle between 0° and 90° with respect to the polarization axis of the source beam. It has been determined that imaging enhancement may be optimized or maximized in embodiments where the QWP 21 is oriented with its fast axis at 45° with respect to the dominant polarization axis of the source beam.

[0063] The portion of the source beam directed into the reference arm 18 by the beam splitter 28 (e.g., an incident portion of a reference beam) is directed to a mirror 40 in the reference arm about a reference path. Mirror 40 functions as a reflector, and reflects the incident beam back into the reference path. The portion of the beam reflected from the mirror 40 (e.g., a reflected portion of the reference beam) is directed back to the beam splitter 28 about the reference path.

[0064] The illustrated embodiments of the reference arm 18 include the QWP 19. As described in greater detail below, the QWP 19 may be positioned or otherwise configured to rotate polarization of the incident and reflected portions of the reference beam by an amount (e.g., a reference path or reference arm amount) between ± 0° - 45° with respect to the rotation produced by the QWP 21 in the sample path (e.g., with respect to the sample arm rotation amount). It has been determined that the imaging enhancement (e.g., reduction of specular reflection from the sample’s surface) may be optimized or maximized in embodiments where the QWP 19 is oriented with its fast axis at ± 45° with respect to the fast axis of the QWP 21 in the sample path (e.g., at 0° or 90° with respect to the dominant polarization axis of the source beam).

[0065] Accordingly, in embodiments, the image enhancement may be optimized by system 10 when the QWP 21 of the sample arm 20 is oriented with its fast axis at 45° with respect to the dominant polarization axis of the source beam, and the QWP 19 of the reference arm 18 is oriented at 0° or 90° with respect to the dominant polarization axis of the source beam. However, other orientations of the QWP 19 and QWP 21 may provide desired or sufficient amounts of image enhancement.

[0066] The reflected portions of the reference and sample beams from the reference arm 18 and sample arm 20 are combined. In the illustrated embodiments of the imaging system 10, the beam splitter 28 combines the reflected portions of the reference and sample beams. The combined reflected portions of the reference and sample beams are then directed (e.g., by optical waveguides) to a spectrometer 50. Spectrometer 50 detects and produces electrical signals representative of the combined reflected beam portions. The electrical signals produced by the spectrometer 50 are coupled to workstation and electronics 52, which processes the electrical signal and produces image information representative of images of three dimensional structures of the sample 44. Other embodiments include other detectors and / or additional collection of the interference signal on the fourth path of the beam splitter 28 (i.e., between the sample beam reflected by the beam splitter and the reference beam transmitted by the beam splitter).

[0067] Spectrometer 50 and workstation and electronics 52 can be conventional or otherwise known systems to provide interference-type imaging information, such as for example optical coherence microscopy or optical coherence tomography. For example the workstation and electronics 52 can operate to provide frequency domain optical coherence tomography or time domain optical coherence tomography. Systems and software for workstation andelectronics 52 are, for example, commercially available. In embodiments, the workstation and electronics 52 can also be used to control the scanner 42. Detectors such as spectrometer 50 are also commercially available.

[0068] With the exception of the QWP 19 and / or QWP 21 as described herein, interferometer 14 may be the same as or similar to conventional or otherwise known interferometers.

[0069] The total electric fields of the partially polarized light source 12 may be considered as being divided into two groups: the linearly polarized electric fields and the nonlinear or unpolarized electric fields. The electric field vector ^^^^( ^^^, ^^) right after the beam splitter propagating in the reference arm 18 (j=r) and sample arm 20 (j=s) of the interferometer14 can be described by Eq. 1 below.^^ ^ ^^ ^^ ^^ ^^^^ ^^ ^^ ^^^( ^^^^, ^^) = ^ ^^൫ ^^ ^^ ^^ି ^^ ^^൯^^ ^^ ^^ ^^ ^^ ^^^ ^^ Eq. 1where ( ^^^^ ^^, ^^^^ ^^) and ( ^^^^the field in the horizontal and vertical axes, respectively, and k=^^ ^^^^ ^^is the wavenumber at the center wavelength. Assuming no material or significant depolarization of the incident light occurs at the sample 44, the electric field ^^ᇱ^^of the reflected / backscattered light in the reference and sample arms 18, 20 after a round trip through the QWP 19 in the sample arm can be expressed by Eq.2 below: ^^^^^^ ^^^ ᇱ ^^ഏା^௭ ା ఏ ି௪௧^^ + ^^ ^^ ^^ ^^మ ೕ ೕ^^^ ^ ^௫^^√ ^^ ^^ ^^ ^^൫ ^^ ^^ ^^൯ା√ ^^ ^^√ ^^ ^^ ^^ ^^൫ ^^ ^^ ^^൯where, ^^ = ^ is the Jones matrix of a QWP with the fast angled at ^^the transposition, ^^^^^^ ^^ ^^ ^^= ^^^^ ^^cos൫ ^^^^ ^^൯ cos൫ ^^ ^^^^൯ + ^^^^ ^^cos൫ ^^^^ ^^൯ sin൫ ^^ ^^^^൯^^^^ ^^ ^^^^= ^^^^ ^^sin൫ ^^^^ ^^൯ cos൫ ^^ ^^^^൯ + ^^^^ ^^sin൫ ^^^^ ^^൯ sin൫ ^^ ^^^^൯ ^^^ ^^ ^^ ^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^interference between the reference and a given layer of the sample 44 can be written as Eq.3 below (the zero-frequency term as well as the self-interference term have been ignored for simplicity):〈 ^^〉 = ∑〈 ^^ᇱ ^^ᇱ∗ + ^^ᇱ∗ ^^ᇱ 〉 = ∑〈2 ^^ᇱ ^^ᇱ ^cos( )^ ᇱ ᇱ^ ^ ^ ^ ^௫ ^௫ ^^^௫ − ^^^௫ + ^^∆ ^^ + 2 ^^ ^௬ ^^ ^௬^cos൫ ^^^௬ −^^^௬ + ^^∆ ^^൯൧〉 Eq. 3where ∆ ^^ = ^^^− ^^^is the optical path length difference between the reference and sample arms, and ^^^௫, ^^^௬, ^^^௫, ^^^௬, ^^ᇱ^௫, ^^ᇱ^௫, ^^ᇱ^௬, ^^ᇱ^௬,are all variables of the summation.

[0072] Using the identities, ∆ ^^௫= ^^^௫− ^^^௫= ^^ ^^௫and ∆ ^^௬= ^^^௬− ^^^௬= ^^ ^^௬, Eq.4〈 ^^〉 = 2∑ 〈 ^^ᇱ^௫^^ᇱ^௫^cos൫( ^^( ^^௫+ ∆ ^^)൯൧ + ^^ᇱ^௬^^ᇱ^௬^cos ^൫ ^^( ^^௬+ ∆ ^^൯^^〉 Eq.5 where ^^௫and ^^௬represent the additional optical path length difference induced by the polarization phase delay between the sample and the reference arms in the x and y axes, respectively.

[0073] The Fourier transformation of Eq.5 can be described as:^^ ^^(〈^^〉)=∑^^ ^^^^^ᇱ^௫ ^^ᇱ^௫^^^^^(^^ ± ( ^^௫ + ∆ ^^))^+∑^^ ^^^ ^^ᇱ^௬ ^^ᇱ^௬൧ ^ ^^൫ ^^ ± ( ^^௬ + ∆ ^^)൯Eq.6

[0074] Note that ^^ ^^^ ^^ᇱ^௫^^ᇱ^௫^ and ^^ ^^^ ^^ᇱ^௬^^ᇱ^௬൧ terms represent different weights of the point spread function (PSF) of the source, and ^ denotes the convolution. Equation 6 indicates that the PSF of the real image is located at ^^௫+ ∆ ^^ and ^^௬+ ∆ ^^ and depends on the polarization of electric fields incident in the reference and sample arms.

[0075] One can consider the three configurations below in connection with the system 10: configuration A, where the sample arm 20 has a QWP 21 with the fast axis of the QWP angled at 45 degrees from the dominant polarization of the source beam and the reference arm 18 has a QWP 19 with the fast axis of the QWP angled at 45 degrees from the dominant polarization of the source beam; configuration B, which is similar to configuration A, but with the fast axis of the QWP 19 in the reference arm 18 angled at 22.5 degrees from the dominant polarization of the source beam; and configuration C, which is similar to configuration A, but with the fast axis of the QWP 19 in the reference arm 18 angled at 0 degree from the dominant polarization of the source beam. These exemplary embodiments A, B and C have the same type of QWP (e.g., QWPs 21, 19) in both the reference and sample arms 18, 20 to balance dispersion.

[0076] In the configuration A where ^^గ^ = ^^^=ସ, Eq.4 leads to ^^௫= ( ^^^௬− ^^^௬) / ^^ and ^^௬= ( ^^^௫− ^^^௫) / ^^, which equal 0 for allincidentthe interference signal is dominated by the contribution of electric fields that are copolarized between the reference and sample arms and the probability of interference between non copolarized electric fields is close to 0.

[0077] In the configuration B where ^^ =గ^^ ^^ ^^ ^^గ^ସ ^=଼, an intermediate case, both copolarized and non copolarized electric fields will contribute to the interference signal.

[0078] In the configuration C where ^^గ^ =ସ^^ ^^ ^^ ^^^= 0, ^^௫= ( ^^^௬− ^^^௫) / ^^ and ^^௬= ( ^^^௫− ^^^௬+ (2 ^^ + 1) ^^)travelling in the sample and reference arms will be orthogonal after the round trip through the QWPs, and therefore will not interfere. The interference signal will be dominated by non colinear electric fields, including circular and elliptical electric fields.

[0079] FIG.2 presents the interference signals and corresponding depth profile of the three configurations while focusing on the front surface of a sample of thin glass of ~150 µm in thickness. FIG.2 includes comparisons of three different arrangements of ^^^for a constant ^^ =గ.గ^ସThe waveform A illustrates an interference signal with ^^^=ସ. The waveform Billustrates an interference signal with ^^^=గ଼. The waveform C illustrates an interference signal with ^^^= 0. Graphs D illustrate the corresponding depth profiles of the three configurations..

[0080] It may be noted in with FIG.2 that the specular reflection from the two highly reflective surfaces of the glass can be reduced to almost 0 by rotating the fast axis of the QWP in the reference arm from ^^⁄4 (A traces) to 0 (B traces). The insert in Fig.2 (D2) exhibits the emergence of two peaks symmetrical around ∆ ^^ and with a respective FWHM that is comparable to that of the configuration A. This may be due to the phase shift between right elliptically polarized and left elliptically polarized electric fields in the reference arm interfering with elliptical electric fields in the sample arm. As the degree of polarization decreases, the probability of interference between nonlinear polarization states increases. This may indicate the optimal degree of polarization of the light source that would yield the greatest effect of this phenomenon, and hence the enhancement of the image contrast.

[0081] Performance of exemplary embodiments of system 10 were evaluated by imaging an infra-red (IR) detector card (VRC5, Thorlabs, Newton, NY). FIG.3 shows the comparison between the three configurations A, B, C. The first row (Figs.3A1, 3B1, and 3C1) represent cross-sectional images of the sample. The second row (Figs.3A2, 3B2, and 3C2) are enface images of the surface. The third row (Figs.3A3, 3B3, and 3C3) are enface images of the sample at the depth location represented by the red arrow Y.

[0082] The configuration A results in a strong specular reflection from the surface of the sample. As ^^^is varied from ^^⁄ 4 to 0, the central peak located at ∆ ^^, corresponding to the surface specular reflection from the interference between colinear polarized electric fields, decreases to become 0 whereas the contribution of unpolarized electric fields becomes significant depending on the degree of polarization of the source. Although the configuration C induces a false double-layer, it does not compromise the axial resolution of the system, thus enables the visualization of tightly packed cellular features. Importantly, the contract of fluorescent particles between the two layers was greatly enhanced in C, compared to A where the visibility was limited.

[0083] Evaluations of prototypes of system 10 were validated by imaging the cornea of a fresh porcine eyeball. FIG.4A-F show the enface images of the different layers of the porcine cornea using the configuration C. FIG 4A shows squamous epithelial cells exhibiting some cells pilling off. FIG.4B shows wing epithelial cells. FIG.4C shows basal epithelial cells. FIG.4Dshows anterior stroma exhibiting corneal verves. FIG.4E shows mid stroma showing keratocytes. FIG.4F shows endothelial cells.

[0084] The above-described results demonstrate that the depth resolution of POCM by embodiments of system 10 (e.g., configuration C) was not or only by relatively limited amounts affected compared with the configuration A. However, the apparition of a duplicated peak (ghost image) may reduce the contrast of individual layers. The contribution of the specular reflection from the surface of the epithelium is greatly diminished to enable the visualization of epithelial cells. Although the use of unpolarized or partially polarized light source has been reported as having a negative effect of the axial resolution of OCT, this feature may be beneficial in certain applications as far as a specific arrangement of the polarization state between both arms of the interferometer is satisfied.

[0085] FIG.5 illustrates an exemplary prototype of POCM system 110 that can be used for in vivo human corneal imaging. Components of system 110 can be similar to those of imaging system 10 described above, and similar features are shown with similar reference nos. The system 110 is powered by a partially polarized near-infrared (NIR) super luminescent diode source 112 (e.g., M-T-850-HP-I, Superlum, Ireland), producing a source beam with a central wavelength of 850 nm and a bandwidth of 165 nm, enabling a theoretical axial resolution of 1.9 µm in air. The partially polarized light source 112 was adopted to enable the evaluation of the polarization-dependent contrast. The source beam produced by light source 112 was transmitted to an interferometer 114 through a 1x2 fiber coupler 116. This common-path approach was adopted to enable control over the polarization in a free space configuration, thus mitigating the need for a broadband polarization-maintained fiber at 850 nm, as well as the system’s instability due to the optical path length shift between the two arms (reference arm 118 and sample arm 120) of the interferometer 114. Other embodiments may include other configurations, such as those that do not include a common-path approach.

[0086] A polarization controller 124 may be integrated into the common-path fiber 126 to adjust the polarization state of the incoming light so that its dominant polarization is horizontal, allowing optimization of the relative contribution of linear and nonlinear polarization portions of the light source 112. A 30:70 (R:T) non-polarizing beam splitter 128 (e.g., BS020, Thorlabs, Newton, NJ) may be used to split the light into the reference and sample arms 118, 120.

[0087] A quarter-wave plate 121 (QWP) (e.g., #46-560, λ / 4 Achromatic Waveplate, Edmund Optics, Barrington, NJ) angled at 45° from the horizontal axis (i.e., dominant polarization) was integrated into the sample arm 120 to turn the horizontal and non-horizontal linear polarizations into circular and elliptical polarizations, respectively. This configuration enables light to penetrate deeper into tissue or other imaged objects or samples. A similar QWP 119 with the fast axis adjustable between 0 and 45° relative to the fast axis of the sample arm QWP 121 was integrated into the reference arm 118 to selectively enable interference of either linear or non-linear polarization states. The dispersion between the two arms 118, 120 may be compensated using a combination of dispersion compensation elements 130 – DCE (e.g., LSM54DC1, Thorlabs, Newton, NJ), a diffraction grating – DG (e.g., GR25-0608, Thorlabs, Newton, NJ), an achromatic doublet – ACD1 (e.g., AC254-030-B, Thorlabs, Newton, NJ), and a flat mirror 140 – M (e.g., PFSQ10-03-P01, Thorlabs, Newton, NJ). The reference arm 118 was mounted on a manual translation stage (e.g., TBB0606, Thorlabs, Newton, NJ), enabling the control of the optical path length difference of the interferometer 114.

[0088] The sample arm 120 included a dual-axis galvanometer scanner 142 (e.g., GVSK2-US, Thorlabs, Newton, NJ) and a 2.5X telescopic relay system using a 30-mm (ACD2) and 75-mm (ACD3) achromatic doublets (e.g., #49-352 and #45-805, Edmund Optics, Barrington, NJ) to conjugate the midway position between the scanning mirrors (distant by 10 mm) with the back focal plane of a microscopic objective 160 and expand the beam so that it overfills the back aperture of the e.g., 10X microscopic objective (e.g., #86-818, Olympus UPLFLN 10x, 0.30 NA, Edmund Optics, Barrington, NJ). The interference signal was recorded with a spectrometer 150 (e.g., Cobra–S 800, Wasatch Photonics, Morrisville, NC), equipped with a 250kHz line-scan camera (e.g., Octoplus 2k, Teledyne e2v, England) and connected to a frame grabber (e.g., AXN-PC2-CL-1xE, Bitflow, Woburn, MA). The synchronization between the scanners and the frame grabber was achieved through a four-channel analog output data acquisition card (e.g., NI USB-6363, National Instruments, Austin, TX). The imaging system 110 was assembled on a 12” x 12” aluminum breadboard that was mounted on a three-axis manual translation stage 162 to enable the alignment of the system with the human eye. As perhaps best shown in FIG.6, the system also integrated a bite bar 170, a headrest 172, and an in-line fixation target 174 to stabilize the participant's head during imaging.

[0089] In connection with a protocol for human corneal imaging, a study of the imaging system 110 included two subjects. A bite bar 170 was made for each subject using the Kerr Impression compound (e.g., Thermoplastic impression material, 1236714, Henry Schein, NY). The mold was set for a few minutes to become rigid before being mounted on the bite bar for POCM imaging. A two-side headrest 172 was integrated into the system 110 to stabilize subjects’ heads during imaging and reduce motion artifacts. For imaging sessions, subjects were comfortably seated on a chair in front of the system and instructed to bite on their bite bar 170. The two-side headrest 172 was placed against the right and left side of the forehead to keep the head as stable as possible during imaging. The headrest 172 was secured using tightening screws without causing discomfort to the subject. Only the one (e.g., the left) eye of each subject was imaged while the other (e.g., right) eye was patched to improve the fixation of the target. The POCM’s scanning head was then moved closer to the subject’s eye (10 mm working distance) using the three-axis manual translation stage 162. A real-time 2D scan display was used to monitor the alignment of the scanning head with the subject’s eye and adjust the plane of focus of the POCM beam. The subject was instructed to fixate on the target during the acquisition of individual volumes. By one exemplary approach, a series of four volumes were collected at each depth of the cornea (e.g., the object being imaged) to increase the likelihood of collecting images free of motion artifacts. The acquisition time of individual volumes was 1 second in examples. Each imaging session lasted for about 30 minutes: 5 minutes for an informed consent, 10 minutes for making the bite bar mold, and 15 minutes for POCM imaging. The instrument 110 operated in a non-contact mode with a 10-mm working distance, thereby limiting any risks of touching the cornea or causing any corneal injuries or infections. The incident power onto the cornea was below 2 mW, which was well below the threshold for thermal tissue damage and in vivo eye safety by ANSI (American National Standards Institute). In addition, the beam focusing onto the cornea will be well spread out when it reaches the retina, further reducing the risk of phototoxicity to the retina. The imaging protocol was similar for both subjects, with the only material or significant difference being the relative orientation of the QWP 119 in the reference arm 118. For the first subject, the fast axis of the reference QWP 119 was set at 45° relative to the fast axis of the sample arm 120 QWP 121 (cross-polarization) whereas it was set at 0° for the second subject (co-polarization).

[0090] In connection with data acquisition and processing, one or more strategies may be used to overcome motion artifacts inherent to in vivo human imaging. By one, for example, the field of view may be reduced (e.g., from 1 x 1 mm2to 0.5 x 0.5 mm2, thus reducing the sampling points from 1000 x 1000 Ascans to 500 x 500 Ascans, yielding a four-time reduction in the acquisition time of individual POCM volumes (from 4 seconds to 1 second)). Although this modification induced a lower signal-to-noise ratio, in examples it may not have demonstrated a significant impact on the image quality. Another exemplary strategy for overcoming the axial motion of the eye includes leveraging the self-interference signal between the corneal surface and underlying corneal layers, enabling high-contrast images of cellular features of the anterior cornea. By this approach, for example, the image is derived from interference between first and second different layers of the sample, where the first layer is optially an outer surface. In embodiments, both images from the self-interference and the interference between the reference arm and the sample were simultaneously captured and distinctly displayed at different depth locations in the same image enabled by the shallow depth of focus of OCM. One benefit of self- interference approaches in POCM is that it enables imaging of the anterior cornea with the highest axial resolution enabled by the source and detector, free of dispersion that may otherwise degrade the axial resolution due to the mismatch of optical components between the reference and sample arms. Conventional or otherwise known or otherwise available programs (e.g., in LabVIEWTM2017 (National Instruments, Austin, TX) and MATLAB® (The Mathworks, Natick, MA) operating in the workstation and electronics 152 were used for data acquisition and processing in connection with these imaging approaches. For example, a flattening algorithm may be applied to data sets to numerically remove the curvature of the cornea and correct for the axial motion artifacts, thus enabling the visualization of cellular features in enface images. The self-interference portion of the images may need no flattening as the corneal curvature and axial motion artifacts were intrinsically compensated. The open-source image-processing platform ImageJ was used for image analysis in embodiments. Other embodiments use other known or otherwise conventional image processing systems.

[0091] In connection with POCM calibration and testing, the effect of polarization and reduced sampling on the resolutions of the system 110 was investigated in embodiments. For purposes of investigation, the axial resolution was evaluated by imaging a thin glass with a thickness of ~150 µm set as the sample. The point spread function (PSF) of the system wasevaluated in three configurations of the relative angle between the fast axes of the reference and sample QWPs 119 and 121 set at 0, 22.5°, and 45°. FIG.10 presents the interference signals and corresponding depth profiles in the three configurations while focusing onto the front surface of thin glass. The specular reflection from the relatively highly reflective front surface of the glass located at ∆ ^^ was reduced by almost 100% by changing the relative angle between the two QWPs 119, 121 from 0° (blue traces) to 45° (green traces). In the latter (45° relatively angle configuration), the polarizations of incident co-linear electric fields in the reference arm 118 and sample arm 120 of the interferometer 114 became orthogonal after the round trip through the QWPs, e.g., assuming no depolarization occurred in the sample, resulting in destructive interference. The image of the sample was therefore formed from the interference between non- linear polarization states, which yielded the emergence of two peaks symmetric around ∆ ^^ (see e.g., the green trace inserted in FIG.10(d2)). This may be due to the phase shift between right elliptically polarized and left elliptically polarized electric fields in the reference arm 118 interfering with elliptical electric fields in the sample arm 120. This false double-peak may cause individual layers to be replicated at a different depth (ghost image), slightly degrading the contrast of individual layers. Nevertheless, the FWHMs of these peaks were comparable to that of the configuration at 0° (FIG.10(d2)) and will produce a similar depth sectioning of the sample 144. The measured axial resolution of 3.2-3.6 µm in air was lower compared to the theoretical value of 1.9 µm, perhaps due at least in part to the chromatic shift of the microscopic objective lens as well as its low transmission in the longer wavelength range. The chromatic dispersion contributed to ~0.4 µm of this axial resolution degradation.

[0092] To evaluate the impact of the reduced sampling points and polarization on the lateral resolution and field of view of POCM, a positive non-fluorescent USAF 1951 resolution test chart (e.g., R1L1S1P, Thorlabs, USA) was imaged in four different configurations, including a combination of 1000 x 1000 A-scans, 500 x 500 A-scans, and relative angle of the reference arm QWP 119 set at 0° and 45°. FIG.7 shows the enface POCM images and line profiles across the 6thelements of the 7thgroup of the test chart in the four configurations. While the definition of the image was better with 1000 sampling points, the system could still resolve the finest elements of the chart with 500 sampling points, which still satisfied the Nyquist sampling requirement over a field of view (FOV) of 0.5 x 0.5 mm2. Thus, this reduced sampling did not affect the lateral resolution in these embodiments of the system. The system operating with 500sampling points was able to resolve the 6th elements of the 7th group, corresponding to a lateral resolution of 2.2 µm, and was adopted for in vivo human imaging. FIG.7a1 represents 1000 x 1000 A-scans with the relative angle of QWPs set at 0⁰, and FIG. 7b1 represents corresponding intensity profiles. FIG.7a2 represents 500 x 500 A-scans with the relative angle of QWPs set at 0⁰, and FIG.7b2 represents corresponding intensity profiles. FIG.7a3 represents 1000 x 1000 A- scans with the relative angle of QWPs set at 45⁰, and FIG.7b3 represents corresponding intensity profiles. FIG.7a4 represents 500 x 500 A-scans with the relative angle of QWPs set at 45⁰, and FIG.7b4 represents corresponding intensity profiles.

[0093] In connection with in vivo imaging of the human cornea, FIG.8 presents cross- sectional and enface images of the cornea of the two subjects evaluated in the above-described study. The cross-sectional images (FIGs.8a1 and 8a2) are for illustration purposes and were not extracted from the same volumes used for enface images. Similarly, enface images were extracted from different POCM volumes as it was desired to focus the beam at different depth of the cornea between acquisitions. The cross-sectional image from Subject 1 captured with the relative angle of 45° between the QWPs exhibits a false double-layer of the ocular surface with an overall low signal (FIG.8a1) whereas the image from Subject 2 captured with the relative angle of 0° between the QWPs exhibits a strong specular reflection from the surface. Both images from the interference between the cornea and the reference arm of POCM (top part of the image) and from the self-interference between the corneal surface and underlying structures (bottom part of the image) are distinctly represented in the images (FIGs.8a1 and 8a2). The self-interference was not affected by the axial motion as long as the amplitude of the motion remained within the depth of focus of the objective lens. Enface images of the corneal surface (FIGs.8b1 and 8b2) exhibit squamous epithelial cells (black arrowhead) as well as hyperreflective features (blue arrows), presumably immune cells or infiltrates from the tear film. While both configurations of the QWPs produced high-contrast images of the stroma (FIGs.8c1 and 8c2), the contrast was reduced with the 45° configuration. Also, the induced double-layer feature may cause an overestimation of the density of keratocytes. Thus, this configuration may be more suitable for imaging the corneal surface, epithelial cells, and endothelial cells.

[0094] FIGs.8a1 and 8a2 are cross-sectional images along the slow-scanning axis of the subjects 1 and 2, respectively, showing axial motion and a blink (gap in FIG.8a2) during the acquisition. FIG.8a1 exhibits a false double-layer of the surface induced by the configurationwith QWPs at 45⁰, whereas FIG.8a2 shows a strong specular reflection from the surface in the configuration with QWPs at 0⁰ (black arrows). The top part of the image corresponds to the interference of the cornea with the reference arm whereas the botom part corresponds to the self- interference, which is not affected by the axial motion. FIGs.8b1 and 8b2 are Enface images of the corneal surface of the subjects 1 and 2, respectively. Hyperreflective features (blue arrows) are visible on the surface, which may be immune cells or infiltrates from the tear film. Low- contrast squamous epithelial cells and their hyporeflective nuclei (black arrowhead) are also visible in FIG.8b. FIGs.8c1 and 8c2 are Enface images of the mid-stroma showing keratocytes (white arrowheads) as well as a nerve branching (green arrow). FIGs.8d1 and 8d2 are Enface images of the endothelium showing endothelial cells.

[0095] FIG.9 shows exemplary enface POCM images of the anterior cornea extracted from the self-interference portion of the volumes (see FIGs.8a1 and 8a2 for illustration). FIGS. 9a1 and 9a2 are Enface images of squamous epithelial cells in both subjects showing some hyporeflective nuclei and cell boundaries. FIGs.9b1 and 9b2 are Enface images of wing epithelial cells in both subjects. FIGs.9c1 and 9c2 are Enface images of basal epithelial cells in the two subjects. FIGs.9d1 and 9d2 are Enface images of sub-basal nerve plexus. FIGs.9e1 and 9e2 are Enface images of the anterior stroma exhibiting keratocytes and a nerve branching in FIG.9e1.

[0096] The disclosed imaging approaches can provide valuable insights into key microstructural feature of imaged samples or objects, such as those of the anterior cornea, and highlight the capabilities of self-interference-based POCM as a powerful tool for non-invasive in vivo imaging (e.g., of the ocular surface). The self-interference portion of POCM images enables delineation between all layers of the anterior cornea with the highest axial resolution, free of chromatic dispersion. Perhaps even more importantly, the self-interference enables issues related to the curvature of the cornea as well as axial motion artifacts that compromised the visualization of cellular features, especially the corneal sub-basal nerve plexus, in a single enface image, to be overcome. These capabilities have important clinical implications for the early detection and management of corneal diseases and may pave the way for numerous longitudinal studies, involving the anterior cornea, such as evaluating the impact of the long-term use of contact lenses on the ocular surface, identifying biomarkers of the early stage of keratoconus as well evaluating the efficacy of corneal collagen cross-linking treatment.

[0097] This technology presents strategies to overcome several shortcomings inherent to cellular resolution in vivo imaging of the human cornea and other structures using SD-OCM. The POCM operated at a fast speed of 1 volume (500 x 500 x 2048 voxels) per second to overcome eye movements and motion artifacts. The integration of a QWP into the sample arm enabled the illumination light to be elliptically polarized, yielding deeper penetration and increased scattering efficiency in tissues. The integration of a QWP with variable orientations into the reference arm enabled different polarization states to contribute or not to the image formation process. This approach was used to mitigate the strong specular reflection from the corneal surface and therefore enhance the contrast of different layers of the human cornea in vivo. While this cross-polarization configuration may not have been optimized to produce images with contrast as high as the co-polarization configuration, the former revealed some benefits of the use of partially polarized light sources in OCT. Although partially polarized light sources have been reported to degrade the axial resolution of OCT, this feature can be beneficial in certain applications given specific arrangements of the polarization states between both arms of the interferometer. As the degree of polarization of the source decreases, the probability of interference between non-linear polarization states would increase and may yield better sensitivity and image contrast. An optimal degree of polarization of the light source may yield the desired image contrast. The sensitivity of the system may be further improved by collecting light from the fourth face of the beam splitter.

[0098] POCM was capable of generating depth-resolved images of all corneal layers, including squamous, wing, and basal epithelial cells, corneal sub-basal nerve plexus, stromal keratocytes, as well as endothelial cells, with lateral and axial resolutions of 2.2 µm and ~2.4 µm in the cornea, respectively, and clarity comparable to IVCM. POCM has an advantage over IVCM of being non-contact and providing high depth-sectioning and real-time visualization of cross-sectional images of the cornea, which can enable to delineate all layers of the cornea, including the tear film.

[0099] Another important advantage of the system described herein is that it enabled high-resolution images of the anterior cornea by using self-interference between the corneal surface and underlying layers. The system can therefore produce non-invasive and non-contact images of the human cornea with the highest axial resolution enabled by the source and detector, free of dispersion mismatch degradation. Furthermore, this approach overcomes two majorchallenges in visualizing cellular microstructures of the cornea: 1) the corneal curvature that generally compromises the visualization of cells in a single enface image and requires to numerically flatten the cornea, and 2) image artifacts due to the axial motion of the eye during the acquisition. This feature offers tremendous research opportunities in the anterior cornea, including 1) understanding the dynamics of epithelial cells in healthy and pathological conditions such as keratoconus, 2) evaluating the interaction between contact lenses, the tear film, and the ocular surface, and 3) evaluating corneal nerves in different disease conditions or the impact of long-term wear of contact lenses on the ocular surface. This approach also provides opportunities for clinical settings to offer ophthalmologists and clinicians the opportunity to thoroughly examine corneal cellular structures with great cellular details and track structural changes associated with disease over time, thus providing valuable information about their pathogenesis and treatment efficacy.

[0100] Further improvements to POCM for clinical settings may include 1) integrating a 2D near-infrared camera to visualize the cornea and enable a fast alignment of the system with the eye, and 2) integrating a three-axis motorized stage into the system for an automatic eye alignment based on the feedback loop from the 2D camera. A cross-correlation approach between successive B-scans of the volume can compensate for any transversal motion artifacts. Although the images reported here were limited to 0.5 x 0.5 mm2to increase the imaging speed and overcome motion artifacts, the system was capable of a FOV of 1.3 x 1.3 mm2, which can be leveraged in applications where a larger FOV is needed. The number of sampling points may then be increased to fulfill the Nyquist sampling requirement and preserve the resolution of the system, which will lower the acquisition speed. The speed of the system can be further improved by upgrading the spectrometer with even faster line-scan cameras or integrating a second spectrometer into the system to enable a tandem acquisition between two spectrometers, further reducing the acquisition time by a factor of at least two to enable motion-artifact-free images of the human cornea. Another strategy for overcoming the speed limitation in SD-OCT is the parallel acquisition of A-scans using line-field SD-OCT or Fourier-domain full-field-OCT.

[0101] Imaging fine structures such as for example those of the cornea may require micrometer-scale axial and lateral resolutions (high N.A. lenses), resulting in a shallow depth of imaging (~ 60 µm for POCM with the use of a 10X microscopic objective lens) and limiting the ability to image the entire corneal thickness in a single shot. Mitigation approaches to extend theimaging depth of POCM may include equipping the system with a dynamic focusing capability, a tunable deformable membrane, using a Bessel beam illumination, or a computational approach. The self-interference approach enabled to overcome the corneal curvature and axial motion artifacts described herein may be used for imaging the anterior portion of the cornea using the corneal surface as a reference. Other embodiments may involve imaging the posterior cornea or any other tissues using the self-interference approach with the endothelium or any other layers of the sample as a reference, which may provide a valuable tool for in vivo investigation of biomarkers of the early stage of FECD.

[0102] In summary, the POCM approaches described herein enable non-invasive and non-contact in vivo imaging of all layers of samples such as those of the human cornea with a cellular-level resolution, which offers tremendous opportunities to evaluate structural alterations that take place in different corneal diseases early in the disease process as well as monitor the disease progression and evaluate treatment efficacy. These POCM approaches have the potential to become a viable clinical tool for ophthalmologists, clinicians, and researchers to thoroughly examine the cornea in normal and pathological conditions.

[0103] Various modifications and additions can be made to the exemplary embodiments discussed without departing from the scope of the present disclosure. For example, while the embodiments described above refer to particular features, the scope of this disclosure also includes embodiments having different combinations of features and embodiments that do not include all of the described features. Accordingly, the scope of the present disclosure is intended to embrace all such alternatives, modifications, and variations as fall within the scope of the claims, together with all equivalents thereof.

Claims

CLAIMS 1. A method for imaging a sample, comprising: providing source light that is partially polarized about a first polarization axis; splitting the source light into a reference beam and a sample beam; scanning an incident portion of the sample beam and directing the scanned incident portion of the sample beam to the sample about a sample path, and causing a reflected portion of the sample beam to be reflected from the sample, including rotating polarization of the incident portion and the reflected portion of the sample beam with respect to the first polarization axis by a sample path rotational amount to reduce reflections from surface portions of the sample that interfere with imaging of subsurface portions of the sample, and wherein the sample path rotational amount is 0° - 90°, and optionally 45°; directing an incident portion of the reference beam to a reflector about a reference path and causing a reflected portion of the reference beam to be reflected from the reflector, including rotating polarization of the incident portion and the reflected portion of the reference beam with respect to the first polarization axis by a reference path rotational amount with respect to the sample path rotational amount, and wherein the reference path rotational amount is ± 0° - 45° with respect to the sample arm rotational amount; combining the reflected portion of the reference beam and the reflected portion of the sample beam to produce a combined beam; and detecting and processing the combined beam to generate information representative of an image of the sample over axial and lateral portions of the sample.

2. The method of claim 1, wherein rotating polarization of the incident portion and the reflected portion of the sample beam includes rotating polarization by an optical component, optionally a quarter wave plate.

3. The method of claim 1, wherein rotating polarization of the incident portion and the reflected portion of the reference beam includes rotating polarization by an optical component, optionally a quarter wave plate.

4. The method of claim 1, wherein the sample path rotational amount is 45°.

5. The method of claim 1, wherein the reference path rotational amount is 45° with respect to the sample path rotational amount (e.g., + 45° or - 45° with respect to the sample path rotational amount).

6. The method of claim 1, wherein the source light is polarized by an amount less than 75 %.

7. The method of claim 1, wherein the source light is polarized by an amount less than 50 %.

8. The method of claim 1, wherein the method includes optical coherence microscopy with and without a reference beam.

9. The method of claim 1, wherein the method includes optical coherence tomography with and without a reference beam.

10. The method of claim 9, wherein the method includes one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

11. The method of claim 1, wherein detecting and processing the combined beam includes generating an image of the sample over axial and lateral portions of the sample.

12. The method of claim 1, wherein detecting and processing the combined beam includes generating an image of the sample over axial and lateral portions of the sample via interference between the reflected portions of the sample beam and the reference beam.

13. The method of claim 1, wherein detecting and processing the portion of the combined beam created by self-interference between any reflected portions of the sample beam and other backscattering beams from the sample to generate an image of the sample over axial and lateral portions of the sample.

14. A method for imaging a sample, comprising: providing source light that is partially polarized about a first polarization axis, including a sample beam; scanning an incident portion of the sample beam and directing the scanned incident portion of the sample beam to the sample about a sample path, and causing a reflected portion of the sample beam to be reflected from the sample, including rotating polarization of the incident portion and the reflected portion of the sample beam with respect to the first polarization axis by a sample path rotational amount to reduce reflections from surface portions of the sample that interfere with imaging of subsurface portions of the sample, and wherein the sample path rotational amount is 0° - 90°, and optionally 45°; and detecting and processing the portion of the combined beam created by the interference between the reflected portions of the sample beam and the reference beam as well as the portion of the combined beam created by the self-interference between different reflected portions the sample beam (from different layers of the sample) to generate information representative of an image of the sample over axial and lateral portions of the sample.

15. The method of claim 14, wherein rotating polarization of the incident portion and the reflected portion of the sample beam includes rotating polarization by an optical component, optionally a quarter wave plate.

16. The method of claim 14, wherein the sample path rotational amount is 45°.

17. The method of claim 14, wherein the source light is polarized by an amount less than 75 %.

18. The method of claim 14, wherein the source light is polarized by an amount less than 50 %.

19. The method of claim 14, wherein the method includes optical coherence microscopy with and without a reference beam.

20. The method of claim 14, wherein the method includes optical coherence tomography with and without a reference beam.

21. The method of claim 20, wherein the method includes one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

22. A system for imaging a sample, comprising: an optical system, including: a light source providing source light partially polarized about a first polarization axis; a sample arm configured to receive a scanned portion of the source light and the sample to be imaged, the sample arm comprising a sample arm quarter wave plate having a fast axis oriented at an angle between 0° and 90°, and optionally 45°, with respect to the first polarization axis of the source light; a reference arm configured to receive a portion of the source light, the reference arm comprising a reference arm quarter wave plate having a fast axis oriented at an angle between ± 0° - 45° with respect to the fast axis of the sample arm quarter wave plate; and a processing system for processing electrical signals corresponding to light received from the reference arm and the sample arm of the optical system, to produce information representative of a three-dimensional image, including depth and transverse information, of the sample.

23. The system of claim 22, wherein the sample arm quarter wave plate has a fast axis oriented at an angle of 45° with respect to the first polarization axis.

24. The system of claim 22, wherein the reference arm quarter wave plate has a fast axis oriented at an angle of + 45° or - 45° with respect to the fast axis of the sample arm quarter wave plate.

25. The system of claim 22, wherein the light source provides source light polarized by an amount less than 75%.

26. The system of claim 22, wherein the light source provides source light polarized by an amount less than 50%.

27. The system of claim 22, wherein the system is configured to provide optical coherence microscopy with and without a reference beam.

28. The system of claim 22, wherein the system is configured to provide optical coherence tomography with and without a reference beam.

29. The system of claim 28, wherein the system is configured to provide one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

30. The system of claim 22, wherein the processing system produces the information representative of the three-dimensional image of the sample due to interference from the light received from the reference arm and the sample arm.

31. The system of claim 22, wherein the processing system produces the information representative of the three-dimensional image of the sample due to self-interference from the light received from different layers of the sample.

32. A system for imaging a sample, comprising: an optical system, including: a light source providing source light partially polarized about a first polarization axis; a sample arm configured to receive a scanned portion of the source light and the sample to be imaged, the sample arm comprising a sample arm quarter wave plate having a fast axis oriented at an angle between 0° and 90°, and optionally 45°, with respect to the first polarization axis of the source light; and a processing system for processing electrical signals corresponding to light received from the sample arm of the optical system via self-interference, to produce information representative of a three-dimensional image, including depth and transverse information, of the sample.

33. The system of claim 32, wherein the sample arm quarter wave plate has a fast axis oriented at an angle of 45° with respect to the first polarization axis.

34. The system of claim 32, wherein the reference arm quarter wave plate has a fast axis oriented at an angle of + 45° or - 45° with respect to the fast axis of the sample arm quarter wave plate.

35. The system of claim 32, wherein the light source provides source light polarized by an amount less than 75%.

36. The system of claim 32, wherein the light source provides source light polarized by an amount less than 50%.

37. The system of claim 32, wherein the system is configured to provide optical coherence microscopy with and without a reference beam.

38. The system of claim 32, wherein the system is configured to provide optical coherence tomography with and without a reference beam.

39. The system of claim 38, wherein the system is configured to provide one or more of frequency domain optical coherence tomography or time domain optical coherence tomography.

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