Quantitative wide-field polarized light microscopy

Abstract

One embodiment of the present invention provides a polarized light microscope (PLM) in an unconventional bistatic configuration that avoids the aberrations that limit the field of view (FOV) of conventional PLMs. One PLM embodiment provides one or more polarization FOVs with an accuracy better than 5% of the total element error of the measured Mueller matrix elements, and the FOV is over 200 times larger than conventional PLMs for both reflection and transmission samples. A further embodiment provides a method for achieving unlimited polarization FOVs for reflection samples by stitching together micrographs acquired as the sample or microscope is translated, without incurring the seam discontinuities that occur when stitching together micrographs of conventional PLMs without digital fusion. Using appropriate models, the embodiments described herein achieve analytical or quantitative wide-field polarized light microscopy applicable to the characterization of material structure, microstructure, and texture, e.g., those related to crystalline structure, and the resulting micrographs generated therefrom.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 393,670, entitled "Quantitative Wide Field Polarized Microscope," filed July 29, 2022, the specification and claims of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable.

[0003] Names of the parties to the joint research agreement Not applicable.

[0004] Incorporation-by-reference of material submitted on a compact disc Not applicable.

[0005] Statement of Prior Disclosure by Inventor or Co-Inventors Not applicable.

[0006] copyrighted work Not applicable.

[0007] Embodiments of the present invention relate to the field of optical microscopy, and more specifically, polarized light microscopy (PLM) for imaging the structure, microstructure, and texture of materials, which may be due to, for example, crystalline structure. One embodiment of the present invention is a laser-based polarized light microscope with a wide field of view (FOV) in terms of resolution and polarization accuracy. Embodiments of the present invention are also in the field of analytical or quantitative microscopy applied to material characterization. Material properties that can be imaged and measured include one or more of the material's chemistry, phase, crystallinity, topography, particle or fiber size and shape, crystal or fiber orientation, stress, and spatial and temporal distribution. A description of the relevant art, including information disclosed under 37 C.F.R. 1.97 and 1.98

[0008] The following description refers to a number of publications by author(s) and publication year, and it should be noted that a particular publication should not be considered prior art to the present invention by virtue of its most recent publication date. Discussion of such publications herein is provided for more complete context and should not be construed as an admission that such publication is prior art for patentability determination purposes. [Background technology]

[0009] Accurate inspection and imaging using polarized light enables a variety of materials characterization techniques, for example, in the fields of crystallography, metallurgy, chiral and polymer analysis, thin films, nanotechnology, and tissue and cell biology. Quantitative if a technique is considered quantitative if the measured polarization feature can be validated against independent measurements or first-principles models, usually by applying a model related to the measured polarization feature to other material features or properties that can be measured by independent instruments.

[0010] Polarization signatures are a measurable combination of optical power, and in the case of imaging, irradiance. In metallography, measured polarized image illumination can be applied to infer crystal orientation through the application of electromechanical models, as demonstrated, for example, in [1], and the measured polarization signatures are validated by comparison of the induced crystal orientation with that measured by electron backscatter diffraction (EBSD) performed in a scanning electron microscope (SEM).

[0011] When applied to characterize microstructures through validated measurements, PLM is called analytical or quantitative polarized light microscopy (qPLM). The techniques applied in qPLM to acquire polarized images of microstructures can also be applied to the polarization imaging of larger objects at longer ranges. Microscopy is a technical field that uses microscopes to observe samples and objects that cannot be seen with the naked eye. While PLM has been utilized to visualize anisotropic microstructures for nearly a century, qPLM is a relatively new technique primarily due to its use of digital image processing, precise polarization metrology, and polarization optics, which can be difficult to acquire and qualify. Few commercially available optical microscopes can achieve qPLM over a field radius greater than a few millimeters. Ellipsometry, on the other hand, has traditionally been quantitative, but primarily extends beyond a range of approximately 1 mm. 2 This limitation makes ellipsometry unsuitable for imaging large areas (e.g., approximately 1 mm) unless multiple images are stitched together to capture a larger imaging area. 2 Super~approx. 225mm 2 Because most ellipsometers employ a broadband white light source and measure polarization intensity at various colors / wavelengths, the optimal optical design and optical components with low polarization aberrations for ellipsometers are different from those for laser PLM microscopes, such as one or more embodiments of the present invention, which measure polarization image irradiance at a single or few wavelengths. 2 An imaging ellipsometer having only a substantially smaller FOV than is demonstrated, for example, in US Pat. No. 6,233,999.

[0012] A microscope image or micrograph is defined by several key parameters, such as spatial resolution, field of view (FOV), and image quality, which can include signal-to-noise ratio (SNR). Accuracy is also very important for quantitative microscopy. For conventional optical microscopes as well as embodiments of the present invention, spatial resolution, image quality, and accuracy all vary with FOV, typically being highest at the center of the image and decreasing at wider field points. The loss of resolution and accuracy with increasing FOV is primarily caused by aberrations, which increase with the field angle relative to the optical axis. A field stop is typically employed to limit the micrograph to an FOV where aberrations are acceptable, and resolution and accuracy are specified as values around this FOV. It is misleading but common to specify the resolution and accuracy of a microscope at the center of a micrograph while specifying a wide FOV. Maintaining polarization accuracy at larger FOVs is particularly difficult, and most commercially available microscopes achieve resolutions on the order of 1 mm. 2 While the polarization FOV is limited to 1000mV, the brightfield / non-polarized FOV can be larger. Conventional PLMs can correct aberrations and achieve polarization accuracy of several mm. 2 Attempts have been made to extend this to larger FOVs, but the results have been inconsistent and are not applicable to the microscope of the present invention, which uses a fundamentally different bistatic configuration.

[0013] Quantitative microscopy requires high absolute accuracy, as defined below for polarimetry, because variations in the material of interest often do not correspond to small changes in reflected or transmitted light. For example, as shown in [Patent Document 1], variations in the crystal orientation of titanium alloys correspond to a maximum variation of less than 5% in polarized reflectance at visible wavelengths. Other materials of interest have smaller anisotropic fractions, resulting in even smaller measurable variations. The accuracy of a polarimeter, which encompasses ellipsometers and PLMs alike, is expressed as the fractional deviation of the Mueller matrix elements of a calibration sample from its theoretical truth, as demonstrated for a non-imaging narrowband laser polarimeter in [Patent Document 2]. Any polarimeter, ellipsometer, or PLM can be mathematically described as a partial Mueller matrix polarimeter (pMMP), as taught in [Patent Document 2]. The accuracy requirements and specifications for a pMMP apply to all measured Mueller matrix elements.

[0014] Wide-area micrographs are necessary for characterizing many materials, industrial components, processes, and faults that exhibit large features or textures, anything over a few millimeters being considered long or large. Furthermore, the finer the texture, the better the benefits from the better statistics provided by wide-area micrographs. For example, anisotropic material properties in crystalline or fibrous materials can extend over areas much larger than the FOV of conventional optical microscopes. Many applications benefit from quantitative wide-field PLM (qwfPLM), as achieved by embodiments of the present invention, which provides better spatial visualization and statistics for applications such as quality control and inspection of high-performance, safety-critical components, e.g., welded and cast aerospace parts.

[0015] Most conventional microscopes are relatively small, typically 100 mm 2 The term "wide field of view" refers to a field of view (FOV) of less than 100 mm, which is not large enough for statistically relevant analysis of many common structures and textures. 2This approach has been applied to a large number of micrographs, each significantly smaller than the original FOV. For such materials, components, and processes, conventional micrographs collected as the sample is translated perpendicular to the optical axis are often stitched together to create a composite micrograph with a larger nominal FOV. While this approach allows for visualization of larger areas, it also tends to emphasize the microscope's limited intrinsic FOV by the appearance of seam discontinuities where individual micrographs are stitched together. Seam discontinuities are conceptually illustrated in Figure 1A, based on PLM crystal images. The microscope's intrinsic FOV corresponds to one square block, 64 of which are stitched together in this example, with different hatching representing different crystal orientations. Seam discontinuities arise from aberrations and other gradients across the intrinsic FOV, such that the right edge ("R") of the first ("primary") image does not coincide with the left edge ("L") of the second ("secondary") image, assuming the sample is translated to the left. Because aberrations generally occur throughout the inherent FOV, not just at the edges, the appearance of high-contrast seam discontinuities typically indicates limited accuracy over a significant portion of the FOV. Image filters or digital fusion routines capable of removing seam discontinuities generally cannot correct for underlying gradients, which have a much greater impact than the edges of the micrograph. While compound micrographs with seam discontinuities may be adequate for quantitative visualization, they may not be precise enough for quantitative microscopy, particularly qPLM; the images are interpreted by a computer and the seam discontinuities may be misinterpreted as features or grain boundaries. Seam discontinuities arise when stitching together conventional bright-field micrographs and are particularly severe in polarized light images, because polarization aberrations are typical but often ignored in the FOV of conventional microscopes (see, e.g., Non-Patent Documents 3 and 4).

[0016] 100mm 2Based on the need for quantitative optical microscopy, particularly qPLM, with sufficient accuracy (described below) over a FOV of >1000 nm, and the inability of existing PLMs to meet these requirements due to their fundamental design geometry, a novel PLM design is needed that achieves higher polarization accuracy over a large FOV. Such a PLM is referred to as a quantitative wide-field PLM (qwfPLM), embodiments of which are further described herein.

[0017] The term "polarization FOV" is introduced to explicitly mean the FOV where a particular polarization accuracy dominates. Polarization accuracy requirements vary depending on the material and application, with better than 5% typically required and better than 1% required for certain materials and applications. Materials with only mild anisotropy, such as unetched martensitic steel, require very high polarization accuracy for satisfactory qPLM.

[0018] The qwfPLM of one embodiment of the present invention can achieve very wide (e.g., 100 mm) axial scanning by placing the objective lens and any other aberrating optical components outside the sample space, as shown in FIG. 2C. 2 This design produces more precise (e.g., better than 1%) polarization imaging over areas (larger than 1000 nm). While this design sacrifices spatial resolution, embodiments of the qwfPLM described herein achieve resolutions of about 5 microns or better for typical reflectance samples, and resolutions of about 1 micron or better are possible for transmission samples and small reflectance samples using a high-resolution attachment according to another embodiment of the invention. Conventional microscopes, on the other hand, sacrifice polarization precision in favor of high spatial resolution. The intrinsic polarization FOV captured by one embodiment of the qwfPLM is over 200 times larger than the polarization FOV captured by a typical commercial product. The FOV generated by one embodiment of the qwfPLM can also be expanded, without fundamental limitations, by stitching together micrographs collected as the microscope sample is translated.

[0019] Another embodiment of the present invention provides an auxiliary sample stage for placement of transmission samples, such as traditional histological and petrographic slides, as well as a method for easily switching between reflection and transmission samples, which is not possible with most commercially available microscopes.

[0020] An embodiment of the present invention provides a 100 mm 2 ~1mm 2 We present a method for generating quantitative PLM (qPLM) and associated micrographs that are smaller than 100 nm. Because the novel microscope experiences negligible polarization gradients across its inherent FOV, its on-axis micrographs can also be more precise than those of conventional PLM.

[0021] Embodiments of the present invention also encompass micrographs and images generated by embodiments of qwfPLM as disclosed herein. Visually or through material and process signature models, these micrographs can reveal material chemistry, phase, crystallinity, topography, particle or fiber size and shape, crystal or fiber orientation, stress, other material or process properties, and their spatial and temporal distribution. [Prior art documents] [Patent documents]

[0022] [Patent Document 1] U.S. Patent No. 7,663,752 [Patent Document 2] U.S. Patent No. 10,540,571 [Non-patent literature]

[0023] [Non-Patent Document 1] "Journal of the Optical Society of America A" 38, 1752 (2021) [Non-patent document 2] "Optics Express" 24, 19881 (2016) [Non-patent document 3] Griffiths et al., “Additive Friction Stir-Enabled Solid-State Additive Manufacturing for the Repair of 7075 Aluminum Alloy,” Appl.Sci.9, 3486 (2019) [Non-patent document 4] H.E. Sims, "Process-Structure-Property Investigation of CP-Ti (Grade 2) Produced via High Deposition AM Laser Hot-Wire," PhD dissertation, Case Western Reserve University (August 2022) Summary of the Invention

[0024] A first embodiment of the present invention is a polarized light microscope (PLM) comprising an electromagnetic radiation (EMR) source (e.g., a narrowband laser) that emits an illumination beam. An image capture device (ICD) (e.g., a digital ICD) is positioned on a bistatic path with the EMR source. For example, the bistatic path includes a bistatic angle of about 5 degrees to less than about 20 degrees. A primary reflection sample plane is positioned at the apex of the bistatic path between the EMR source and the ICD. A sample space is bounded by the primary reflection sample plane and includes adjacent, continuous portions of the bistatic path. A polarization state generator is positioned outside the sample space, within the path of the illumination beam, between the EMR source and the primary reflection sample plane. The illumination beam follows the bistatic path. A polarization state analyzer is positioned outside the sample space, on the bistatic path, between the primary reflection sample plane and the ICD. The objective lens is positioned outside the sample space, on the bistatic path, between the polarization state analyzer and the image capture device, and the objective lens forms an image of the primary reflection sample plane at an image plane coincident with the image capture device. For example, in one embodiment, the objective lens is the only lens between the primary reflection sample plane and the image capture device. In a further example, the PLM is positioned approximately 5 mm 2 Over 300mm 2has an instantaneous field of view (FOV) of less than about 100 mm, for example, 2 ~about 300mm 2 In a further embodiment, the illumination beam is about 100 mm 2 To achieve an instantaneous FOV of 1000 or greater, the PLM has a diameter of about 20-50 mm or greater. In one embodiment of the present invention, the PLM has an accuracy of about 5% or less, or about 2% or less, or about 1% or less of total element error in the measured partial Mueller matrix of the calibration mirror at all pixels within the FOV that meet the ICD functional pixel specifications. The PLM of this first embodiment may further include one or more of: 1) a sample translation element that moves the sample parallel to the primary reflective sample plane for imaging; 2) a calibration mirror positioned coincident with the primary reflective sample plane; and 3) an auxiliary transmission sample stage positioned in sample space between the primary reflective sample plane and the PSA, constructed with a flat, aberration-free window, and including a transmission sample plane; and / or 3) a high-resolution attachment positioned in sample space between the calibration mirror positioned coincident with the primary reflective sample plane and the PSA, constructed with a flat, aberration-free mirror, and including a secondary reflective sample plane.

[0025] A second embodiment of the present invention is a 2 ~about 300mm 2 , for example, about 100 mm 2 Over or about 300 mm 2 A second embodiment provides a method for generating a polarized image (e.g., a micrograph) of a sample area less than about 50 mm, the method comprising imaging the sample with a PLM (e.g., the PLM of the first embodiment), wherein the sample area image is a single captured polarized image generated without stitching together smaller images. 2 By stitching together multiple adjacent polarized images of 2 The invention may further include generating a stitched polarization image of greater than about 50 mm. For example, stitching multiple adjacent polarization images together produces an image with no or minimal seam discontinuities without the use of digital fusion. In a further embodiment, the invention may further include generating a stitched polarization image of greater than about 50 mm. 2Stitching together multiple adjacent polarization images of over 50 mm results from moving the sample relative to a fixed position on the PLM and / or 2 Multiple adjacent polarization images of the sample result from moving the PLM relative to the sample, which is in a fixed position.

[0026] A third embodiment of the present invention provides a photomicrograph of a material sample imaged with a polarized light microscope (PLM) (e.g., the PLM of the first embodiment), wherein the photomicrograph is an image of approximately 100 mm of the material sample generated without stitching together smaller images. 2 ~about 300mm 2 Provide a micrograph, which is a single micrograph of an area. For example, a PLM is approximately 50 mm 2 ~about 300mm 2 In one embodiment, the micrograph does not contain seam discontinuities and the micrograph is not stitched from multiple images.

[0027] A further scope of the applicability of the present invention will be set forth in part in the following detailed description taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. [Brief explanation of the drawings]

[0028] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate one or more embodiments of the invention and, together with the description, serve to explain the principles of the invention. The drawings are only for purposes of illustrating one or more embodiments of the invention and are not to be construed as limiting the invention. In the drawings:

[0029] [Figure 1A] 1 shows an illustration of a stitched crystal image produced by conventional PLM exhibiting seam discontinuities (known in the art). [Figure 1B]Figure 1B shows a diagram of a stitched crystal image produced by conventional PLM exhibiting seam discontinuities (known in the art). Figure 1B shows a diagram of a grain boundary map produced from the stitched image of Figure 1A.

[0030] [Figure 1C] FIG. 1B is an illustration of a grain boundary map generated from PLM according to an embodiment of the present invention, with a larger intrinsic polarization FOV compared to the conventional PLM used to acquire the crystal image and the grain boundary map generated in FIGS. 1A-B.

[0031] [Figure 2A] 1 is a schematic diagram of the optical configuration of a conventional monostatic reflectance microscope.

[0032] [Figure 2B] 1 shows a conventional monostatic reflective PLM.

[0033] [Figure 2C] 1 illustrates a bistatic reflective PLM according to one embodiment of the present invention.

[0034] [Figure 3] FIG. 1 is a diagram of a qwfPLM according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] Conventional reflected light microscopes utilize a monostatic normal incidence design, as shown in Figure 2A. Referring to Figure 2A, a conventional reflected light microscope includes an electromagnetic radiation (EMR) source 101 that emits a beam that is directed toward an objective lens 103 and focused onto a sample surface 104 before returning to a beam splitter and being detected by a camera 102. This monostatic design allows the objective lens to be positioned very close to the sample, thereby achieving high magnification and on-axis spatial resolution.

[0036] Referring to Figure 2B, a conventional PLM is shown in which the objective lens 103 is located between two polarization modulators 105 and 106 in the sample space 107. The first polarization modulator 105, following the light source, is a polarization state generator ("PSG"), and the second polarization modulator 106, following the sample, is a polarization state analyzer ("PSA"). A beam splitter 109 is also necessarily located in the sample space 107 of a monostatic PLM. Despite sophisticated and careful manufacturing, it is difficult to achieve a monostatic PLM, especially for 100mm PLMs. 2 For FOVs approaching 1000 nm, it remains very challenging to fabricate beamsplitters and objective lenses with ultra-low polarization aberrations. In addition to inherent imperfections, small levels of mechanical stress and thermal gradients can cause polarization aberrations in these optical components.

[0037] One embodiment of the present invention is a qwfPLM with a bistatic design, as shown in FIG. 2C . An EMR source 101 generates a beam (e.g., but not limited to, visible light) that is directed through a PSG 105 to a primary reflection sample plane 104. The PSG 105 is located outside the sample space 107. The reflected EMR propagates from the sample plane 104 to a PSA 106, which is located outside the sample space 107. The EMR propagates through an objective lens 108 to an image capture device 111, and the sample image plane 110 corresponds to the imaging plane of the image capture device. This bistatic design does not require a beam splitter (not present and therefore not shown) and places the objective lens 108 outside the sample space 107. Thus, the embodiment shown in FIG. 2C avoids aberrations from the beam splitter and / or objective lens of a conventional microscope. In one embodiment, the image capture device 111 of the embodiment of FIG. 2C may be tilted to coincide with the sample image plane 110, which may be inverted relative to the actual sample plane 104. For example, as described in "Optics Express" 24, 19881 (2016), using polarization optics pre-qualified by a separate metrology polarimeter to ensure that the polarization optics meet the requirement of less than 1% total element error in the Mueller matrix for the design shown in FIG. 2C can be achieved in a 100 mm 2The qwfPLM provides accuracy of at least a few percent, preferably better than 1%, over a substantially larger polarization FOV. According to one embodiment, the qwfPLM of the present invention employs a narrowband laser light source to avoid chromatic polarization variations and aberrations. This embodiment does not use a white light source or a monochromator. Furthermore, another embodiment uses only a single lens between the sample and the image capture device. Because the PLM of the present invention does not use a beam splitter, there is less light loss, resulting in the utilization of a lower-power EMR source compared to conventional PLMs employing beam splitters. As used herein, a sample can be a user-defined sample or a calibration mirror.

[0038] Other existing microscopes based on bistatic geometry, broadly referred to as "oblique illumination microscopy," have not been designed or demonstrated for quantitative PLM. Oblique illumination is often used as a form of dark-field microscopy to enhance the contrast of transparent samples or features. Combining images recorded under different oblique illuminations can also improve spatial resolution, but these techniques still suffer from polarization aberrations when the objective and / or other optical components are located within the sample space. For example, light-sheet or selective plane illumination microscopy, such as that described in U.S. Pat. No. 8,582,203, is often based on fluorescent dyes and employs bistatic geometry to achieve tomographic imaging of thick samples, but is not designed for quantitative PLM. Some bistatic qPLMs that retain the objective within the sample space have been demonstrated to limit their FOV. A typical polarimetric microscope, as demonstrated in Applied Optics 45 (22), 5479 (2006) and described in a non-imaging form in U.S. Pat. No. 5,956,147, holds an objective lens in the sample space and has a polarized FOV of 100 mm. 2The microscope's FOV is further limited by the intentional dispersion of the illuminating and reflected beams on the objective lens. The bistatic polarimeter described in U.S. Patent Application Publication No. 2020 / 0271911 similarly limits its polarization FOV by positioning the objective lens (or "electromagnetic radiation collector") between the sample and the PSA (or "second polarization modulator"). Even if the objective lens is placed on the opposite side of the PSA, the transparent aperture of the PSA is still less than 100 mm. 2 If the FOV is less than 100 mm, it does not increase the FOV of these PLMs, as is the case with commercially available photoelastic modulator (PEM)-based PSAs. Other common ellipsometers have been demonstrated for imaging, and are called "imaging ellipsometers," as described, for example, in U.S. Patent No. 7,663,752, but their FOV is less than 100 mm. 2 , which is limited in this case by the large bistatic angles retained from conventional ellipsometry, typically greater than 50 degrees.

[0039] Referring to Figure 3, a top view of one embodiment of a qwfPLM is shown, according to one embodiment of the present invention. The qwfPLM comprises an EMR source (1), preferably a narrowband laser (1), positioned in a bistatic path with an image detector or image capture device (2), with a primary reflection sample plane (3) disposed therebetween. The bistatic angle is typically small, e.g., 8 degrees, but can be smaller or larger depending on the size of the optical components and the polarization effects being measured. The qwfPLM further comprises expansion and collimation optics (4) that generate an expanded, nearly collimated beam (5) that illuminates the sample. The illumination beam can have a diameter of 20-50 mm or smaller to achieve a wide intrinsic FOV. A larger effective FOV is achieved by translating the sample under the illumination beam, employing a translation stage (6) on the sample assembly, and stitching the resulting images together using digital image processing routines. Due to its low-aberration design, the qwfPLM achieves stitched images with negligible seam discontinuities without the use of digital fusion routines. In another embodiment of the invention, suitable for use on stationary samples such as live welds, stitched micrographs are created by translating the entire microscope parallel to the sample surface on a precision dolly.

[0040] If the reflective sample is highly polished (like a mirror) or metal polished, the angle of incidence on the sample is half the bistatic angle, as shown in the qwfPLM embodiment of FIG. 3. Another embodiment of the invention can be applied to rougher samples that are diffusely reflective, in which case the angle of incidence can be variable relative to the bistatic angle. For non-specular imaging of diffuse reflectors, the image capture device is tilted to ensure it remains parallel to the image plane. In another embodiment, the microscope can be positioned to image a transmission sample by mounting a calibration mirror coincident with the primary reflection sample plane (3) and mounting the transmission sample on an auxiliary / transmission sample holder / stage (7) in the sample space near the PSA (6).

[0041] By placing the objective lens outside the sample space, the qwfPLM of the present invention sacrifices resolution to achieve more precise polarization imaging over a very large area. Conventional microscopes, on the other hand, sacrifice polarization accuracy in favor of high spatial resolution. Despite stitching, the resolution of an embodiment of the qwfPLM is approximately 5 microns across the entire FOV, which is fine enough for many applications. For applications requiring finer resolution, another embodiment of the present invention includes a high-resolution attachment placed in the position of the auxiliary stage (7) shown in FIG. 3. For small reflective samples, the high-resolution attachment can achieve resolution down to approximately 2 microns, and in some cases, submicron resolution. Resolutions better than approximately 2 microns are also achievable for transparent samples of a size that does not obscure the illumination beam when placed on the auxiliary stage. In one embodiment, the area of the auxiliary stage and high-resolution attachment through which the reflected EMR passes can be constructed with a flat, non-aberrated mirror or window.

[0042] The qwfPLM further comprises a first independent polarization modulator (8) embedded in the polarization state generator (PSG) and configured to sequentially modulate the polarization state of the probe beam among a set of independent polarization states. The invention further comprises a second polarization modulator (9) embedded in the polarization state analyzer (PSA) and mechanically independent from the first polarization modulator, followed by an objective lens (10). The transparent apertures of the PSA and objective lens are large enough to allow the required imaging resolution and FOV. The polarization modulator can be one of several established devices for UV, visible, or IR light, such as a polarizing crystal, wave plate, or sheet mounted on a manual or preferably motorized rotary stage, or a sequence of such components, or two or more non-rotating polarizing components mounted on a wheel or sliding linear stage, or a registered channel multiplexer (RCM) such as that described in U.S. Pat. No. 10,540,571, which allows high-speed imaging up to video rates. In one or more embodiments, the polarization modulator is not a photoelastic modulator (PEM) or sequence thereof. The combined configuration of the first and second polarization modulators is time-multiplexed to define multiple independent, adjustable polarization channels. An image capture device ("ICD") (2), preferably a CCD or CMOS focal plane array (FPA), is positioned to receive light from the objective lens, and the image capture device generates a set of pixelated signals or images synchronized with the set of channels formed by the PSG and PSA. Embodiments of the qwfPLM may further include a processor (11) connected to and in communication with one or more memories (12) to collect and store raw images from at least the image capture device and / or transmit the raw images to storage. The processor and memory may be on-board the qwfPLM or remote from the qwfPLM and may communicate wirelessly or via a wired connection. The image capture device may also include a large number of pixels, for example, about 16,000 pixels, or about 16,000 to about 1,000,000 pixels, or more than about 1,000,000 pixels, for example, 50,000,000 pixels or more. A suitable ICD provides for the use of a percentage of non-functional (dead and hot) pixels.The large number of pixels allows for a wide range of magnifications of the sample. In one embodiment of the present invention, the optical magnification is approximately 1 in order to avoid aberrations.

[0043] The qwfPLM according to one embodiment of the present invention is otherwise based on established optical designs utilizing commercially available or custom lenses and mirrors, most of which are polarization-maintaining or pre-calibrated to eliminate systematic measurement errors. In particular, pre-calibration standards and associated corrections, as applied to polarization modulator components, characterize another embodiment of the present invention that is more likely to achieve quantitative PLM.

[0044] Using a certified commercially available polarization modulator, the intrinsic FOV of one embodiment of the qwfPLM is 225 mm 2 The FOV can be expanded without fundamental limitations by stitching together micrographs collected as the sample is translated perpendicular to the bisector (horizontally in Figure 2C) or by translating the microscope on a precision dolly. The low-aberration design ensures that these composite images do not suffer from seam discontinuities.

[0045] Seam discontinuities that appear in compound micrographs formed by stitching images together as the sample is translated can be applied to quantify the precision and quantitative FOV of a microscope. Figure 1A shows stitched crystal images generated using a conventional PLM exhibiting seam discontinuities. The microscope's native FOV corresponds to one square block, 64 of which are stitched together in this example, with different hatching representing different crystal orientations. The seam spacing indicates that the native FOV of this PLM is 225 mm, the native FOV of one embodiment of the qwfPLM of the present invention. 2 Much smaller than about 1mm 2This suggests that conventional PLM micrographs are often applied for particle counting, where any substantial change in crystal orientation is interpreted as a grain boundary (GB). For images with seam discontinuities, such as those in FIG. 1A, the estimated number of particles is higher than the actual number of particles because, in addition to actual GBs, some of the seams have false GBs, as shown in FIG. 1B. One embodiment of the qwfPLM of the present invention, due to its low-aberration design, provides a much larger intrinsic (also referred to herein as instantaneous) FOV, enabling more precise particle counting over a much larger area than conventional PLMs, as shown in FIG. 1C. The qwfPLM of the present invention eliminates or minimizes polarization aberrations, providing a significantly larger intrinsic FOV and eliminating or reducing to negligible levels seam discontinuities in the stitched image.

[0046] As used herein, "a," "an," "the," and "said" mean one or more unless the context of the statement dictates otherwise.

[0047] In at least one embodiment, as will be readily appreciated by those skilled in the art, an apparatus according to the present invention includes a general or general-purpose computer or distributed system programmed with computer software to perform the above-described steps, which may be in any suitable computer language, such as C++, FORTRAN, BASIC, Java, assembly language, microcode, distributed programming languages, etc. The apparatus may also include multiple such computers / distributed systems (e.g., connected by the Internet and / or one or more intranets) in various hardware implementations. For example, data processing may be performed by a suitably programmed microprocessor, computing cloud, application-specific integrated circuit (ASIC), field-programmable gate array (FPGA), etc., in conjunction with appropriate memory, network, and bus elements.

[0048] It should be noted that in the specification and claims, "about" or "approximately" means within twenty percent (20%) of the referenced quantity. All computer software disclosed herein may be embodied on any computer-readable medium (including combinations of media), including without limitation CD-ROM, DVD-ROM, hard drive (local or network storage device), USB key, other removable drive, ROM, and firmware.

[0049] Although the invention has been described in detail with particular reference to these embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art, and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above are incorporated herein by reference.

Claims

1. A polarized light microscope (PLM), comprising: an electromagnetic radiation (EMR) source that emits a beam of radiation; an image capture device positioned in a bistatic path with the EMR source; a primary reflecting sample plane positioned at the apex of the bistatic path between the EMR source and the image capture device; a sample space bounded by the primary reflecting sample plane and including adjacent, continuous portions of the bistatic path; a polarization state generator positioned outside the sample space, in the path of the illumination beam, between the EMR source and the primary reflection sample plane; a polarization state analyzer positioned on the bistatic path between the primary reflection sample plane and the image capture device and outside the sample space; an objective lens positioned on the bistatic path between the polarization state analyzer and the image capture device, outside the sample space, and forming an image of the primary reflected sample plane at an image plane coincident with the image capture device; PLM equipped with.

2. The PLM is approximately 5 mm ２ Over 300mm ２ 10. The PLM of claim 1 having an instantaneous field of view (FOV) of less than 100 .mu.m.

3. The PLM is approximately 100 mm ２ ~Approx. 300mm ２ 2. The PLM of claim 1, having an instantaneous FOV of

4. 10. The PLM of claim 1, wherein the PLM has an accuracy of about 5% or less total element error in a measured partial Mueller matrix of a calibration mirror.

5. The PLM of claim 1 , wherein the EMR source is a narrowband laser.

6. The radiation beam has a width of about 100 mm ２ 3. The PLM of claim 2, having a diameter of about 20-50 mm or greater to achieve said instantaneous FOV or greater.

7. The PLM of claim 1 , wherein the bistatic path includes a bistatic angle of about 5 degrees to less than about 20 degrees.

8. 10. The PLM of claim 1, further comprising a sample translation element that moves the sample parallel to the primary reflection sample plane for imaging.

9. The PLM of claim 1 , wherein the image capture device is a digital image capture device.

10. The PLM of claim 1 , wherein the objective lens is the only lens between the primary reflection sample plane and the image capture device.

11. 10. The PLM of claim 1, wherein the primary reflection sample plane is at an arbitrary angle relative to the illumination beam, and the image capture device is appropriately angled to coincide with the image plane.

12. 2. The PLM of claim 1, further comprising: a calibration mirror positioned coincident with the primary reflective sample plane; and an auxiliary transmission sample stage positioned in the sample space between the primary reflective sample plane and the PSA, the auxiliary transmission sample stage being constructed with a flat, non-aberration window and including a transmission sample plane.

13. The PLM of claim 1 further comprising: a calibration mirror positioned coincident with the primary reflective sample plane; and a high-resolution attachment positioned in the sample space between the primary reflective sample plane and the PSA, the high-resolution attachment being constructed of a flat, non-aberrated mirror and including a secondary reflective sample plane.

14. Approximately 50 mm ２ ~Approx. 300mm ２ 1. A method for producing a polarized light image of a sample area, comprising: imaging the sample with a PLM, the sample area being imaged being about 50 mm ２ ~Approx. 300mm ２ wherein the single captured polarized image is generated without stitching together smaller images of the sample area.

15. The method of claim 14, wherein the PLM is the PLM of claim 1.

16. The sample area captured in a single polarized image is approximately 300 mm ２ The method of claim 14, wherein

17. The sample area captured in a single polarized image is approximately 100 mm ２ The method of claim 14, wherein the

18. Approximately 50 mm ２ By stitching together multiple adjacent single captured polarization images of over 100 mm, ２ The method of claim 14 , further comprising generating a super-stitched polarization image.

19. 20. The method of claim 18, wherein the multiple adjacent single captured polarization images are stitched together to produce an image with no or minimal seam discontinuities without the use of digital fusion.

20. Approximately 50 mm ２ 20. The method of claim 18, wherein the plurality of adjacent single captured polarization images result from moving the sample relative to a fixed position of the PLM.

21. Approximately 50 mm ２ 20. The method of claim 18, wherein stitching together the plurality of adjacent single captured polarization images results from moving the PLM relative to the sample, the sample being in a fixed position.

22. The method of claim 14 , wherein the polarized image is a photomicrograph.

23. 2. A photomicrograph of a material sample imaged with the polarizing microscope of claim 1, comprising:

10. A photomicrograph produced by the polarized light microscope of claim 1, wherein the photomicrograph is produced from an image of about 100 mm of the material sample without stitching together smaller images. ２ ~Approx. 300mm ２ A photomicrograph is a single micrograph of an area of.

24. The PLM of claim 1 is about 50 mm ２ ~Approx. 300mm ２ 24. The photomicrograph of claim 23, having an instantaneous FOV of

25. 24. The photomicrograph of claim 23, wherein the photomicrograph does not include a seam discontinuity.

26. 24. The photomicrograph of claim 23, wherein the photomicrograph is not stitched from multiple images.

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