The invention relates to imaging systems which are capable of detecting features of a medium which are responsive to polarized light.
Confocal microscopy is a well-established technology with sub-micrometer lateral (perpendicular to the optical axis) and micrometer longitudinal (parallel to the optical axis) resolution. In a typical biomedical setting, this provides optical images of sections of tissue for qualitative and quantitative cellular morphology, pathology, and chemical analysis. The contrast and resolution of these images allows them to be compared to the gold standard of histopathological preparation and viewing of sectioned and stained tissue.
The use of Nomarski techniques applied to confocal microscopy, and especially laser scanning confocal microscopy, is known to enhance the contrast of objects with phase variations or surface profile variations. Such differential interference contrast (“DIC”) microscopes split a uniform, linearly-polarized pupil such that two point spread functions form at the focus of the objective. This is accomplished using a birefringent prism, such as a Nomarski or Wollaston prism, placed at the pupil (or conjugate location to the pupil) of the microscope. The prism shears the beam into two beams and the orthogonally polarized pupils of the objective are focused to form two telecentric (in the object space) polarized spots. Upon reflection from the object, the sheared polarized beams are collected by the objective and re-combined at the pupil. Passing the recombined beam through a polarizing element provides an interference image, which is based on the phase profile of the scanned sample.
Such DIC configurations were previously improved by circularly-polarizing the sheared beams in order to further enhance the resulting image by reducing interference from turbidity above and below the section being imaged. See, for example, U.S. Pat. No. 6,577,394 to Zavislan, titled “Imaging System Using Polarization Effects to Enhance Image Quality.” FIG. 1 of the Zavislan patent depicts a prior art configuration of the polarization optics and objective of a microscope using a birefringement prism to shear a linearly polarized beam into two linearly polarized beams (having polarization orthogonal to each other) and a quarter wave plate retarder to circularly polarize the beams (opposite-handed polarization states).
By illuminating the sample with sheared beams having generally circular polarization in opposite senses (left and right handed circular polarization), images obtained using light returned from the image plane (i.e., a section within the sample), which may be altered by the sample's circular dichroism, retardation, etc., have reduced image distortion, such as that caused by scattering sites adjacent to the image plane or section.
In biological and tissue objects, there may also be polarization information in linear birefringence, linear di-attenuation, circular dichroism, etc. This information can supplement the morphology and reflectance data that has otherwise been captured. Many methods for gaining polarization sensitivity with a microscope have been investigated. Confocal microscopes have been designed to detect the light's state of polarization after reflection from the object. Although prior techniques have improved the usefulness of images produced, some image information related to polarization was ignored and the detection pathways of such instruments were generally treated as ellipsometers.
While previous efforts have utilized polarized-light techniques for reducing noise in a generated image, none have taken further advantage of the returned differential information to enhance images with details of Polarization Sensitive Features of the object being imaged. Accordingly, it is an objective of the present invention to provide improved imaging systems, such as, for example, imaging systems using confocal microscopy, laser scanning confocal microscopy, scanning reflectance confocal microscopy, etc, that can generated images having information related to the Polarization Sensitive Features of an object. The disclosed apparatus and methods are configured to measure the irradiance of light returned from the object across the lateral (with respect to the optical axis) dimension.
For a fuller understanding of the nature and objects of the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which:
The present invention may be embodied as an apparatus 10 for imaging a sample of a medium 90, which may be a turbid sample. The apparatus 10 may be, for example, a scanning reflectance confocal microscope (“SRCM”), such as that depicted in
In such an apparatus 10, light is received at the medium 90 and illuminates a section of the medium 90. Reflected light is returned from the section and from sites adjacent to the section. The apparatus 10 comprises an optical system 12 for directing light to the medium 90 (“received light”) and directing light from the medium 90 (“returned light”). In some embodiments, the light directed to the medium 90 may be laser light provided by a single spatial mode laser 92.
The optical system 12 is configured to shear the illuminating beam provided by the laser 92 into two beams of differing polarization as is known in the art. For example, in some embodiments, the light from the laser 92 is linearly polarized and passes through polarization processing optics 32. Referring back to the exemplary prior art embodiment depicted in
The beams A, B are directed through an objective 30 and are focused in the medium 90 at spots C, D which are spaced from each other in the focal plane (in the image section of interest) and/or along the optical axis (not shown). In some embodiments of the present invention, the location for the prism 42 is the aperture stop of the objective or at pupil of the optical system. The beams A, B generally overlap outside of the focal region such that both beams illuminate the noise-producing scatterers outside (above and below) the focal region. Because the overlapping region above and below the focal region are illuminated by orthogonally polarized beams, light scattered from isolated scatters outside the focal region is partially canceled by destructive interference. Consequently, any collected signal is reduced by the interference of the light returned from the scattering sites outside of the focal region. However, circular dichroism, optical retardance, di-attenuation and other optical activity also exist and may manifest as differences between the light returned from the spots C, D. Thus, this imaging mode provides a preferential and differential polarization sensitive signal from the focal region. The polarization sensitive signal at the focal region is not strongly influenced be polarization properties of the object above it as is the case in polarization sensitive optical coherence tomography. (J. de Boer et al, Two-dimensional birefringence imaging in biological tissue by polarization-sensitive optical coherence tomography, Optics Letters, Vol. 22, Issue 12, pp. 934-936 (1997) http://dx.doi.org/10.1364/OL.22.000934). Thus, this imaging mode provides the preferential ability to measure the polarization properties of isolated structures within an object.
The light is returned from the medium 90 and collected by the objective 30. The polarization components of each of the angularly sheared return beams that are orthogonally polarized to the original incident sheared beams are not recombined into a single beam, but instead are re-sheared by the prism 42. The optical system 12 of an apparatus 10 of the present invention further comprises a pinhole lens assembly 16 configured to receive the returned light. The pinhole lens assembly 16 is located after a beamsplitter 20 such that returned light is directed to the pinhole assembly 16. In such embodiments of NR-DIC operation, the ensemble of the DIC prism plus quarter-wave-plate is operated between crossed linear polarizers; a linear analyzer is used prior to the pinhole lens that is crossed relative to the initial laser polarization. This is be done, for example, by using a beam splitter 20 that transmits linear polarization of one orientation and reflects the orthogonal linear polarization. Such a beamsplitter is termed a polarizing beamsplitter (“PBS”).
Alternatively, as shown in
It should be noted that if the beamsplitter 20 is an NPBS, a mechanically-adjustable waveplate 149, such as, for example, a Babinet-Soliel retarder, or an electrical adjustable waveplate, such as, for example, a voltage controlled liquid-crystal retarder, can be placed after the NPBS and prior to the analyzer. The retardation axis of the adjustable waveplate may be oriented at 45 degrees. This adjustable retarder can combined with a linear analyzer 150 to provide for a fully ellipsometric analysis of the two return light beams and the light in the area of overlap between the two beams.
Because the prism 42 re-shears light that is orthogonally polarized as it traverses the prism 42 toward the pinhole 16 and recombines the light coincidentally polarized, the spatial distribution of the light in the pinhole 16 depends on the modification of the incident polarization by the object 90. The polarization modifications of interest are, again, linear birefringence, di-attenuation (linear dichroism) and circular dichroism.
The apparatus 10 comprises a detector 60 configured to receive the returned light from the pinhole lens assembly 16. The detector 60 has at least two sensors 61, each sensor 61 configured to receive a portion of the returned light. Each sensor 61 may be, for example, a photo diode. For example, two sensors may be configured as a split photodetector and each sensor 61 may be configured to receive a portion of the returned light that corresponds to one of the beams of light. In another example, a detector has three sensors, two of which are configured to receive a portion of the returned beams and the third being configured to receive a portion of the returned light between the positions of the two beams or wherein the beams overlap.
In another example, a detector has four sensors, the outer two elements of which are configured to receive a portion of the returned beams, and the center two elements are configured to receive a portion of the returned light between the positions of the two beams or wherein the beams overlap. Such a detector configuration 161 (as viewed from the detector lens) is shown in
In yet another example of a detector suitable for the disclosed apparatus, the detector may have an array of sensors, such as an avalanche photodiode array, a charge coupled device (“CCD”), or complementary metal-oxide-semiconductor (“CMOS”) image sensor. The size and spacing of the sensor elements can be varied across the detector array to optimize the filling of each of the sensor elements with each beam or the area overlap between the beams. In this way, the apparatus 10 is configured to detect the spatial distribution of light received at the focus of the detector lens and from the spatial distribution detect the differences in polarization response of the section of the medium 90. In other words, the detector differentiates the light returned from the Polarization Sensitive Features of the medium 90. Additional types of “split detectors” (detectors having more than one sensor) may be suitable for use with the present disclosure.
In one embodiment, the pinhole lens assembly comprises a detector lens that images the spots C and D at or within the object to a pinhole that acts as a spatial filter. The size of the pinhole is typically expressed in relationship to the size of a diffraction limited spot that would be formed at the focus of the detector if the object were replaced by a mirror, placed at the beam waist of the incident illumination. A pinhole aperture of diameter equal to one diffraction limited spot presented at the focus of the detector lens is termed a one-resolution element or one resolution pinhole. Typically, pinhole diameters of one to nine resolution elements are used in laser scanning confocal microscopes. Because the prism 42 produces two sheared spots at the object, the size of one resolution element at the pinhole is increased by the amount of shear scaled by the optical magnification between the objective lens focus and detector lens focus.
Detector elements can be placed behind the pinhole and within one depth of focus of the detector lens, for example, as illustrated in
In another embodiment, the detector lens focuses light onto a sensor array without a pinhole, for example, as shown in
In yet another embodiment, a fiber optic array can be placed at the focus of the detector lens to collect the spatial distribution of light. Individual fibers 380 in an array 382 can be routed to individual detectors 61 as shown in
In another embodiment, a segmented optic can be placed at the focus of the detector lens or at a relayed image behind a pinhole to collect the spatial distribution of light and distribute it to a collection of individual detectors. An exemplary geometry of a segmented optic and its detectors is shown in
Each sensor 61 of the detector 60 produces an electrical signal in response to the portion of the returned light received by the sensor 61 from the pinhole assembly 16. The electrical signal of the detector 60 varies according to characteristics of the light received at the detector 60. The amplitude of the electrical signal may be considered to be generally proportional to the reflectance of the section. In some embodiments, the electrical signal may vary according to a polarization parameter of the received light, such as, for example, the amount and orientation of linear birefringence, the amount of linear dichroism, or di-attenuation, and/or the amount of circular dichroism within the section.
The apparatus may further comprise a processor 62 in communication with the sensors 61 of the detector 60. The processor 62 is programmed to generate an image of the section based on the electrical signal of the sensors 61. The generated image may include image information of the Polarization Sensitive Features. The medium 90 can be scanned by the apparatus in any manner. In the exemplary embodiment depicted in
The present invention may also be embodied as a method 100 of imaging a section of a medium, the section having Polarization Sensitive Features (see
The light incident on the medium is returned by the section (at an image plane) and also from sites adjacent to the section. The light returned from the medium is directed 106 to a detector by way of a birefringent component, such as a prism, wherein polarization components the sheared beams are not recombined. In this way, the beams of light reaching the detector are differentiated according to each beam's light returned from the Polarization Sensitive Features. The direction 106 may be provided by, for example, an optical assembly as described above. The method 100 comprises the step of detecting 109 the differentiation of the beams of returned light received at the detector. For example, the returned light at more than one lateral position of the returned light (with respect to the optical axis) may be detected by a different sensor of the detector such that the detector can sense the differences across the returned light. The sensors may be configured to detecting portions of returned light having the response of a Polarization Sensitive Feature, the sensors may be configured to detect portions having overlapping responses, or the sensors may be configured differently (such that some detect overlapping responses and others do not).
An image of the section is generated 112 from the detected 109 returned light. The generated 112 image may correspond to a polarization parameter of the returned light and include information of the Polarization Sensitive Features. The polarization parameter may be any characteristic of interest to the operator. For example, in some embodiments, the polarization parameter is the amount and orientation of linear birefringence within the section. In other embodiments, the polarization parameter is the amount of linear dichroism within the section. In other embodiments, the polarization parameter is the amount of circular dichroism within the section. Embodiments may include more than one type of response.
It should be noted that the benefit of providing NR-DIC optics along with configuring the sensors elements to detect portions of returned light having the response of a Polarization Sensitive Feature can be applied to optical coherence tomography (“OCT”) imaging systems. OCT systems provide images within tissue by collecting the light scattered from the tissue and interfering it with light from a reference arm. Optical coherence tomography systems are known (D. Huang, et al. “Optical coherence tomography, Science vol. 254, pgs. 1178-1181, 1991; J. M. Schmitt, A. R. Knuettel, A. H. Gandjbakhche, R. F. Bonner, “Optical characterization of dense tissues using low-coherence interferometry”, SPIE Proceedings, vol. 1889 pgs 197-211, July 1993; Handbook of Optical Coherence Tomography, B. Bouma and G. J. Tearney, eds, Markel Dekker, NY (2002) ISBN 0-8247-0558-0; M. Choma, M. Sarunic, C. Yang, and J. Izatt, Sensitivity advantage of swept source and Fourier domain optical coherence tomography, Optics Express, Vol. 11, Issue 18, pp. 2183-2189 (2003), http://dx.doi.org/10.1364/OE.11.002183). OCT systems use time domain, Fourier domain, and swept wavelength source methods to provide interference-based detection as described in Bouma and Tearney (2002) and by Choma et al. (2003). Images can be acquired by: (1) mechanically translating the tissue relative to the optical system; (2) mechanically translating the complete optical system or just the objective relative to the tissue; (3) optically scanning the object illumination beam relative to the optical axis of the objective; (4) imaging the object on to a one-dimensional or two-dimensional detector array; or a combination of (1), (2), (3) and/or (4). Systems that optically scan the object illumination beam are sometimes referred to as optical coherence microscopes (H. Wang, J. A. Izatt and M. D. KulKarni, “Optical Coherence Microscopy” chapter 10 (pgs. 275-298) and H Saint-Jalmes, et al. “Full-field optical coherence microscopy” chapter 11, (pgs. 299-334) Handbook of Optical Coherence Tomography, B. Bouma and G. J. Tearney, eds, Markel Dekker, NY (2002) ISBN 0-8247-0558-0) and can provide images with lateral resolution comparable to confocal microscopy.
Polarization sensitive OCT imaging systems have been developed. (J. de Boer et al, Two-dimensional birefringence imaging in biological tissue by polarization-sensitive optical coherence tomography, Optics Letters, Vol. 22, Issue 12, pp. 934-936 (1997) http://dx.doi.org/10.1364/OL.22.000934). These systems do not utilize the NR-DIC assembly of prism 42 and waveplate 44. Additionally, such previous systems do not utilize the spatial distribution of the light at the detector lens that is returning from the object.
The improved imaging provided by NR-DIC with spatial detection can be incorporated both in scanning spot or scanned object OCT or OCM as well as full-field OCM systems.
Within the object arm, the collimated light is directed through a beamsplitter 120 and into a NR-DIC system comprising a Nomarski or Wollaston prism followed by a quarter-wave plate placed at either the aperture stop of the objective or at a pupil of the objective. In one embodiment, the beamsplitter 120 is a polarizing beamsplitter. Because these systems use broadband light, the quarter-wave plate, beamsplitters, and fiber splitters used in OCT or OCM systems may be designed for the wavelength range used. The NR-DIC assembly produces two sheared, orthogonally circularly polarized beams that are focused to two sheared beam waists within the sample 190. Light scattered within the tissue is collected by the objective and is retransmitted through the NR-DIC system. Light orthogonally polarized to the incident light will be reflected by the beamsplitter where it is focused through a detector lens onto a four-element fiber bundle 384. The fibers of the fiber bundle are oriented so that a line connecting the centers of the fibers is parallel with a line connecting the images of spot C and D. Each fiber collects a portion of the spatial light distribution at the focus of the detector lens. Each of the four fibers from the object arm are combined with one of the four reference arm fibers with a two to one fiber coupler 2. Light from each of the sampled spatial areas in object arm 112 mixes with light from the reference arm 132 by way of the fiber splitter to enable an interference signal that is detected by one of four detectors 160. Thus, the amplitude and phase of each sampled spatial area can be detected using standard OCT or OCM reconstructions by a processor 162. From this reconstructed signal, polarization sensitive features can be determined by the processor 162 or by a separate processor 163.
Fiber-based or free space polarization rotators or analyzers may be incorporated in the source, object, reference, and detection fibers to enable balanced detection. The specific detector and detection algorithm depends on the type of OCT: time domain, Fourier domain, or swept wavelength source. The detector is interfaced with a processor 162 that extracts the information associated with the object being imaged. The processor 162 may be interfaced to an additional processor 163 that controls the translation of the scanning system of the optical system or tissue as well as providing the necessary control signals to the reference arm and the illumination source. Processor 163 may have the ability to display, store, and/or transmit images.
The optical arrangement of the reference arm is similar to the object arm. Light directed to the reference arm is transmitted through an NR-DIC assembly to produce two sheared, orthogonally circularly polarized fields incident on reference mirror 250. The shear prism in the NR-DIC assembly may be placed at the aperture stop of the objective 231 or at a pupil of the reference arm. For each point on the reference mirror being imaged, there are two orthogonally circularly polarized beams that overlap at each reference mirror point. In the preferred embodiment, the mirror surface being imaged is located nominally at the rear focal point of the reference arm objective. Light scattered from each point at the reference mirror is collected by the objective 231, passed through the NR-DIC optics, and directed toward an area detector 260 through the non-polarizing beam splitter and detector lens 212. Light from the object arm 212 is mixed with light from the reference arm 232 by the beamsplitter 220 to enable an interference signal that is detected by a detector 260. A linear polarization analyzer may be placed in the detection arm and the azimuth of the analyzer adjusted to balance the detection of the object and reference arms. In an embodiment, the detector 260 is placed at the rear focal point of the detector lens. The NR-DIC optics in both the reference arm and object arm are placed at aperture stop of the respective objectives or at a pupil of the objectives. The NR-DIC and objective assemblies of both the object and reference arms are positioned such that the pupils of both the reference and object arms coincide with the front focal point of the detector lens. To extract information associated with object, the reference arm may be phase modulated. In an embodiment, the reference mirror is moved in steps of λ0/4 where λ0 is the mean wavelength of the illumination spectrum normalized by the detector responsivity. Irradiance from the detector is captured at three or more measurements taken at consecutive mirror motion steps and processed to extract the phase and amplitude of the light scattered from a section located in the front focal point of the object arm objective. The processing follows that of known phase extraction techniques (J. C. Wyant, Computerized interferometric measurement of surface microstructure, SPIE Proceedings vol. 2576, pgs 122-130 (1996)). The detector is interfaced with a processor 262 that extracts the information associated with the object being imaged. The processor 262 may be interfaced to an additional processor 263 that controls the translation of the optical system to select the depth of imaging (z) within the tissue or the specific (x,y) location of the tissue as well as providing the necessary control signals to the reference arm mirror translator and the illumination source. Processor 263 preferably has the ability to display, store and transmit images.
In embodiments where the detector 260 is an area detector such as a CMOS or CCD imaging array, the magnification and numerical aperture (NA) of the objective may be matched with the pixel size and spacing of the array, such that there are at least two pixel elements across each optical resolution element or resolution at the detector. In some embodiments, rectangular pixel elements may be used, such as those found in some linear CMOS or CCD imaging sensor arrays. These arrays could be used to create an area image by optically or mechanically scanning to create a two dimensional image.
Discussion of Mechanism
The following discussion is intended to be a non-limiting illustration of a mechanism by which the present invention operates. In some embodiments of NR-DIC operation, the ensemble of the DIC prism plus quarter-wave-plate is operated between crossed linear polarizers; a linear analyzer is used prior to the pinhole lens that is crossed relative to the initial laser polarization. This can be done by using a beam splitter 20 that transmits linear polarization of one orientation and reflects the orthogonal linear polarization. Such a beamsplitter is termed a polarizing beamsplitter (“PBS”). Alternatively, as shown in
It should be noted that if the beamsplitter 20 is an NPBS, a mechanically-adjustable waveplate 149, such as, for example, a Babinet-Soliel retarder, or an electrical adjustable waveplate, such as, for example, a voltage controlled liquid-crystal retarder, can be placed after the NPBS and prior to the analyzer. The retardation axis of the adjustable waveplate may be oriented at 45 degrees. This adjustable retarder can combined with a linear analyzer 150 to provide for a fully ellipsometric analysis of the two return light beams and the light in the area of overlap between the two beams.
In the embodiments having a crossed linear analyzer, the orthogonal, sheared polarization states produce projections along the analyzer direction that are 180-degrees out of phase. In the following analysis, the x direction is parallel to the line connecting the spots C and D. The y direction is perpendicular to the line connecting the spots C and D. The z direction is locally parallel to the optical axis of the objective lens and detector lens. The focusing optical elements are assumed to be centered and rotational symmetric. This coordinate system is generally rotated about the z-axis 45 degrees from that shown in
The expected polarization response of a system using NR-DIC techniques can be calculated by tracing its polarization properties in a formalism known as Jones calculus. Any coherent, fully-polarized beam can be decomposed into components of orthogonal polarization. The state of polarization can be represented by a vector containing the magnitude and phase of these two orthogonally polarization states,
where |Ux| and |Uy| are the magnitudes of the x- and y-polarized components and φx and φy their respective phases. The input polarization of our SRCM is x-polarized. This can be represented by the normalized vector
Modifications to the polarization state by optical elements or a polarization sensitive object can be represented by a 2×2 Jones matrix, M, and the output polarization state follows the linear algebra calculation:
U
out
=M·U
in. (3)
The first relevant optical element that the light encounters in the NR-DIC configuration is the birefringent prism that angularly shears the orthogonal polarizations of the illumination. The two states that emerge from the NR-DIC polarization can be represented as ±45° rotations of the input x-polarized beam,
where the rotation matrix R (θ) is
Note, translating a Nomarksi prism laterally across the optical axis of the incident beam adds an average phase bias, δbias, between the polarization states that leave the prism. Note also that alternate adjustable waveplates can be added to bias the phase with either a Nomarski prism or a Wollaston prism. For example, a liquid crystal waveplate can be used to vary the bias under electrical control. The phase bias is represented by the exponential scalars of ±δbias/2. What are left are two orthogonal linear polarization states oriented at ±45° to the input x-polarized beam
These two polarization states will be operated on by the quarter-wave retarder (oriented with its optical axis along the x-axis), modified by the polarization dependent properties, if any, at the object and traced back through the quarter-wave retarder to the prism. The ensemble of these polarization operations can be collapsed to an equivalent Jones matrix
M
System
=M
QWP
·M
Object
·M
QWP. (7)
A coordinate system convention is used herein in which a reflection from a uniform surface at the object plane is identical to the identity matrix, I. In this convention, the orientation of the optical elements does not reverse for light returning from the object surface. The components of the Jones matrix MSystem in our coordinate system are
The object's polarization response is embedded in MObject. The object can selectively attenuate one polarization projection with MAttn or it can add a phase difference between the polarization projections with MPhase. These modifications are a function of the orientation of the object's optical axis, and in general are mathematically:
where P(2θObject) is called the psuedo-rotation matrix. It is a rotation matrix to account for an arbitrary rotation of the objects polarization axis relative to the global coordinate system and is defined as
The terms of note within these expressions that govern the polarization response of the object are the extinction ratio of any di-attenuation present, √{square root over (η)}, and the phase delay of any linear-birefringence present, δobject.
The linear dichroism or di-attenuation variable, η, is defined on the range from 0 to 1, with 0 representing complete di-attenuation (perfect linear polarizer) and 1 representing no di-attenuation.
The linear birefringence parameter, δobject, represents the accumulated phase difference between a field along the material's optical axis and orthogonal to that axis. For an index-of-refraction difference, Δn, between the fields along the optical axis and orthogonal to that axis, the accumulated phase δobject=2πΔn/λ.
The polarization vectors reflected from the object that represent the two angularly sheared polarization states prior to the return trip through the prism are
U
L=αLMsystem·U+45
U
R=αRMsystem·U−45. (11)
Note, the effect of any circular dichroism can also be modeled if a scalar constant (αL,R) representing the amount of relative absorption of either the left- or right-circular polarization states at the object is prepended to the appropriate sheared polarization state.
The choice of L and R subscripts are used for sheared polarization vectors which represent light that was left and right circularly polarized in object space, and result in left and right oriented PSFs at the pinhole plane.
If a completely homogenous, perfectly reflecting, non-polarizing object is placed into the Jones calculus, the matrix representing the entire system response for the NR-DIC system MSystem is the product of the two quarter-wave rotators, MSystem=MQWP·MQWP. This is identically a half-wave rotator and will rotate the +45° oriented polarization vector to −45° and vice versa for the −45° vector. For these flipped polarization states, the return trip through the DIC prism results in a re-shearing of the component beams. That is, they now have twice the angular shear as they did in the illumination direction. Light that is polarization modified by the object, will have some component that does not get angularly re-sheared, but becomes co-linear with the optical axis; this is what occurs in a standard DIC microscope operating with linear input polarization states. Because of this a-priori knowledge of the redirection properties, the light's return trip through the DIC prism after reflection at the object can be thought of as an analysis by ±45° linear polarizers. Each of the two sheared components returning to the prism are analyzed by each of these DIC prism analyzers. This determines their respective angular direction, and thus their respective position in the pinhole plane of the SRCM. As a result, we have 4 field components
M
A,±45 represent linear analyzers oriented at ±45°. The U′L and U′R field vectors are the components that are angularly re-sheared, and U′C1 and U′C2 are the components that are not re-sheared, but are directed back to the optical axis of the SRCM; the C subscript denotes a center position. The bias translation of the prism is also included. The U′L and U′R field vectors cancel their bias terms as they pick up an the complex conjugate of the phase in their illumination directions. The bias effect on the center components is to double the amount of bias-phase from the illumination direction. Since the center components are colinear along the optical axis, their fields are added
Standard operation of the NR-DIC mode is under a linear analyzer oriented at 90° to the initial x-linear polarization, MA,90, prior to the pinhole plane. The fields in the left, right, and center positions in the pinhole are then
U
L,pin
=M
A,90
·U′
L
U
R,pin
=M
A,90
·U′
R
U
C,pin
=M
A,90
·U′
R (14)
The shear specifications of the prism chosen to operate the SRCM will govern the overlap of the three PSFs. With no polarization modification by the object, there is no energy in the center distribution, UC. This is the case of a specular object. Using the Jones calculus for the NR-DIC system, the effect of a polarization modification at the object on the pinhole distribution can now be modeled.
Linear Birefringence
Linear birefringence is the differential optical path that light having two orthogonal linear polarizations encounters as it traverses a medium. This occurs because the index-of-refraction of the material is anisotropic (but still homogenous within some region). For convenience only, and not intended to be a limitation on the present disclosure, this discussion and analysis will be restricted to materials that exhibit anisotropy along one axis—so-called “uni-axial materials.” Such materials have a characteristic optical-axis, which is the axis in which the index-of-refraction is different from the other two. The effect of this physical property on the light is to retard or advance the phase of the light's electric field that lies along this optical axis. A common biological material that is known to exhibit birefringence is collagen.
For a linearly-birefringent object, the evolution of the point spread function (“PSF”) at the pinhole using NR-DIC microscopy (wherein the return beams are not entirely recombined) is shown in
Because the polarization properties of the object vary the distribution of the light in the pinhole along the shear direction, the split-detection structures and methods of the present disclosure can be used to detect such properties. A normalized difference of the integrated irradiance across respective halves of the pinhole along the direction of shear will be used as a metric.
This signal is dependent on the angle of the object's optical axis and the strength of its phase difference. For a given amount of phase birefringence, the maximum of this metric will occur when the object's birefringence axis is inclined at 45 degrees to the horizontal or equivalently, along the shear direction. Oriented at this angle, the resultant Ssplit, for increasing phase birefringence is plotted (
Circular Dichroism
Circular dichroism also affects the pinhole signal with this split geometry. Circular dichroism is the differential absorption of left- or right-circular polarized light. The coefficient αL(x,y) and αR(x,y) represent the amount of relative absorption of either the left- or right-circular polarization states, respectively, at an object point (x,y) within the section being imaged. The NR-DIC mode can have both left and circular polarization states incident on the object. These left and right circular states are correlated to the left and right PSFs in the pinhole plane. Differential absorption of one of these states will lead to a biasing effect similar to that of linear birefringence. However, the biasing does not add light to the overlap region; the biasing is caused by the reduce beam irradiance that is a direct result of the decreased reflectance for one of the circular polarizations. Illustrated in
Di-Attentuation
A linear dichroism or di-attenuation signal also affects the NR-DIC pinhole signal and irradiance. Di-attenuation is the difference in the absorption/attenuation of electric field along one axis. The strength of this attenuation is characterized by the extinction ratio, η. Di-attenuation is commonly found in sheet polarizers that preferentially absorb one linear polarization state and transmit the orthogonal polarization. In the present discussion, a reflective geometry is considered where light that is back scattered from an object contributes to the signal. In this geometry, di-attenuation refers to the preferential absorption of one electric field component relative to another field component in the backscatter collection geometry; the non-absorbed component has enhanced contribution. The di-attenuation variable, η, is defined on the range from 0 to 1, with 0 representing complete di-attenuation (perfect linear polarizer) and 1 representing no di-attenuation. For this type of polarization effect, the total pinhole irradiance is affected but no left/right biasing occurs. The pinhole evolution for increasing extinction ratio is illustrated in
It is possible to extract polarization features from split detectors metrics as described above for each point in the object imaged. Two detectors do not provide sufficient information to differentiate circular dichroism and linear birefringence and cannot differentiate di-attenuation from a reduction in overall reflectance. A three detector system comprising a left detector labeled “L,” a center detector labeled “C,” and a right detector labeled “R,” provides the ability to differentiate circular dichroism and linear birefringence and can differentiate di-attenuation from a reduction in overall reflectance. One possible signal construct for a three-detector system is:
where α is calibration factor that can be measured from the image of a uniform isotropic surface object such as a glass interface to normalize the S′III parameter to zero in the absence of a polarization based object parameter. Another polarization specific parameter would be to detect a modified split detector metric:
Still another polarization specific parameter would be to detect:
S′″III=L+R−ΔC
where Δ is calibration factor that can be measured from the image of a uniform isotropic surface object such as a glass interface to normalize the S′″III parameter to zero in the absence of a polarization based object parameter. Such a system has the ability collect information from each imaged point in an object to provide an image that is related to the total backscatter collected: SIII=L+R+C as well as a polarization sensitive signals S′III, S′III and S′″III.
Considering a four element detector as shown in
where β is calibration factor that can be measured from the image of a uniform isotropic surface object such as a glass interface to normalize the S′IV parameter to zero in the absence of a polarization based object parameter. Another polarization specific parameter would be to detect a modified split detector metric:
Another polarization specific parameter would be to detect a modified split detector metric: S′IV=L+R−ρ(LC+RC), where ρ is calibration factor that can be measured from the image of a uniform isotropic surface object such as a glass interface to normalize the S′″IV parameter to zero in the absence of a polarization based object parameter. Such a system has the ability collect information from each imaged point in an object to provide an image that is related to the total backscatter collected: SIVL+R+LC+RC as well as a polarization sensitive signals S′IV, S″IV and S′″IV.
It is noted that the polarization specific features shift the mathematical moments of the irradiance distribution at the focus of the detector lens. Therefore, polarization information can be extracted by estimating the one-dimensional moments normalized by the number of elements of the irradiance distribution for each imaged point in an object. Consider an n-element sensor where n is even. The optical axis is centered between the n/2 and n/2+1 elements. Moments m=0, 1, . . . n/2 can be calculated. The mth moment Im is:
where Si is the ith detector element. Next consider an n-element sensor where n is odd. The optical axis is centered on the n/2+1 element. Moments m=0, 1, . . . (n−1)/2 can be calculated. The mth moment Im is:
where Si is the ith detector element. For all n-element sensors the integrated signal
can be obtained for each point in the object.
We note that all the parameters mentioned for two or more sensor element detector that sample the irradiance distribution may be biased by changing phase bias of the Nomarski prism or by utilizing full ellipsometric detection enabled by using a non-polarizating beam splitter combined with a waveplate compensator and adjustable analyzer. Adjusting of the phase bias of the Normarski prism or that of the compensator and/or angle of the analyzer may be done to capture different signatures in success images to elucidate the polarization properties of the object.
Although the present invention has been described with respect to one or more particular embodiments, it will be understood that other embodiments of the present invention may be made without departing from the spirit and scope of the present invention. Hence, the present invention is deemed limited only by the appended claims and the reasonable interpretation thereof.
This application claims priority to the provisional patent application entitled “Device and Method for Detection of Polarization Features,” filed Mar. 15, 2013 and assigned U.S. App. No. 61/793,921, the disclosure of which is hereby incorporated by reference.
This invention was made with government support under contract numbers 5R42CA110226 and 5T32AR007472 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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61793921 | Mar 2013 | US |