Polarization microscope using a near infrared full-Stokes imaging polarimeter Wei-Liang Hsu,1,* Jeffrey Davis,1 Kaushik Balakrishnan,1 Mohammed Ibn-Elhaj,2 Shona Kroto,3 Neal Brock,3 and Stanley Pau1 1

University of Arizona, College of Optical Sciences, 1630 East University Boulevard, Tucson, Arizona 85721, USA 2 Rolic Technologies Ltd., CH-4123 Allschwil, Switzerland 3 4D Technology, 3280 East Hemisphere Loop #146, Tucson, Arizona 85706, USA * [email protected]

Abstract: This paper presents a polarization microscope using an infrared (IR) full-Stokes imaging polarimeter. The IR polarimeter utilizes an optimized interference-based micropolarizer design, and provides fullStokes images with resolution of 1608 × 1208 at 35 frames/second. The device fabrication, instrument calibration, performance evaluation, and measurement results are presented. The measurement error of the imaging polarimeter is less than 3.5%, and the standard deviations are less than 2%. ©2015 Optical Society of America OCIS codes: (160.3710) Liquid crystals; (160.5470) Polymers; (130.5440) Polarizationselective devices; (260.5430) Polarization; (180.0180) Microscopy.

References and links 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

R. Oldenbourg, Biomedical optical phase microscopy and nanoscopy (Elsevier, 2013), 311–338. A. E. Tuer, S. Krouglov, N. Prent, R. Cisek, D. Sandkuijl, K. Yasufuku, B. C. Wilson, and V. Barzda, “Nonlinear optical properties of type I collagen fibers studied by polarization dependent second harmonic generation microscopy,” J. Phys. Chem. B 115(44), 12759–12769 (2011). G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, and M.-C. Schanne-Klein, “In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy,” Biomed. Opt. Express 3(1), 1–15 (2012). V. J. Pansare, S. Hejazi, W. J. Faenza, and R. K. Prud’homme, “Review of long-wavelength optical and NIR imaging materials: contrast agents, fluorophores, and multifunctional nano carriers,” Chem. Mater. 24(5), 812– 827 (2012). Y. Shimotsuma, M. Sakakura, P. G. Kazansky, M. Beresna, J. Qiu, K. Miura, and K. Hirao, “Ultrafast manipulation of self-assembled form birefringence in glass,” Adv. Mater. 22(36), 4039–4043 (2010). R. Oldenbourg, “Polarization microscopy with the LC-PolScope,” https://darchive.mblwhoilibrary.org/handle/1912/6277 (2003). C. R. Carey, T. LeBel, D. Crisostomo, J. Giblin, M. Kuno, and G. V. Hartland, “Imaging and absolute extinction cross-section measurements of nanorods and nanowires through polarization modulation microscopy,” J. Phys. Chem. C 114(38), 16029–16036 (2010). G. Myhre, W.-L. Hsu, A. Peinado, C. LaCasse, N. Brock, R. A. Chipman, and S. Pau, “Liquid crystal polymer full-stokes division of focal plane polarimeter,” Opt. Express 20(25), 27393–27409 (2012). P. Taroni, A. Pifferi, A. Torricelli, D. Comelli, and R. Cubeddu, “In vivo absorption and scattering spectroscopy of biological tissues,” Photochem. Photobiol. Sci. 2(2), 124–129 (2003). X. Huang, I. H. El-Sayed, W. Qian, and M. A. El-Sayed, “Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods,” J. Am. Chem. Soc. 128(6), 2115–2120 (2006). M. L. Schroeter, M. M. Bücheler, K. Müller, K. Uludağ, H. Obrig, G. Lohmann, M. Tittgemeyer, A. Villringer, and D. Y. von Cramon, “Towards a standard analysis for functional near-infrared imaging,” Neuroimage 21(1), 283–290 (2004). TruesenseImaging, KAI-2020 image sensor device performance specification (TruesenseImaging, 2012), 17–17. W.-L. Hsu, K. Balakrishnan, M. Ibn-Elhaj, and S. Pau, “Infrared liquid crystal polymer micropolarizer,” Appl. Opt. 53(23), 5252–5258 (2014). W.-L. Hsu, J. Ma, G. Myhre, K. Balakrishnan, and S. Pau, “Patterned cholesteric liquid crystal polymer film,” J. Opt. Soc. Am. A 30(2), 252–258 (2013). G. Myhre, A. Sayyad, and S. Pau, “Patterned color liquid crystal polymer polarizers,” Opt. Express 18(26), 27777–27786 (2010). J. S. Tyo, “Design of optimal polarimeters: maximization of signal-to-noise ratio and minimization of systematic error,” Appl. Opt. 41(4), 619–630 (2002). D. S. Sabatke, M. R. Descour, E. L. Dereniak, W. C. Sweatt, S. A. Kemme, and G. S. Phipps, “Optimization of retardance for a complete Stokes polarimeter,” Opt. Lett. 25(11), 802–804 (2000).

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18. J. L. Pezzaniti and R. Chipman, “Mueller matrix imaging polarimetry,” Opt. Eng. 34(6), 1558 (1995). 19. S.-Y. Lu and R. A. Chipman, “Interpretation of Mueller matrices based on polar decomposition,” J. Opt. Soc. Am. A 13(5), 1106 (1996). 20. R. Chipman, “Polarimetry,” in OSA Handbook of Optics (McGraw-Hill, 1995). 21. C. F. LaCasse, R. A. Chipman, and J. S. Tyo, “Band limited data reconstruction in modulated polarimeters,” Opt. Express 19(16), 14976–14989 (2011). 22. D. H. Goldstein, “Polarization properties of Scarabaeidae,” Appl. Opt. 45(30), 7944–7950 (2006). 23. H. Arwin, R. Magnusson, J. Landin, and K. Järrendahl, “Chirality-induced polarization effects in the cuticle of scarab beetles: 100 years after Michelson,” Philos. Mag. 92(12), 1583–1599 (2012). 24. S. Gao and V. Gruev, “Bilinear and bicubic interpolation methods for division of focal plane polarimeters,” Opt. Express 19(27), 26161–26173 (2011). 25. S. Gao and V. Gruev, “Gradient-based interpolation method for division-of-focal-plane polarimeters,” Opt. Express 21(1), 1137–1151 (2013). 26. D. A. LeMaster and S. C. Cain, “Multichannel blind deconvolution of polarimetric imagery,” J. Opt. Soc. Am. A 25(9), 2170–2176 (2008). 27. X. Zhao, X. Pan, X. Fan, P. Xu, A. Bermak, and V. G. Chigrinov, “Patterned dual-layer achromatic microquarter-wave-retarder array for active polarization imaging,” Opt. Express 22(7), 8024–8034 (2014).

1. Introduction A conventional microscope measures the panchromatic images and provides valuable information of light intensity as well as wavelength (color). Polarization is a third attribute of the optical field which can also provide a large amount of important information. Polarization microscopy has been extensively studied over the past decades [1]. While qualitative polarized light microscopy renders polarized objects in vivid colors, quantitative microscopic polarization images, such as birefringence and dichroism maps, can be obtained using a polarimeter as an imaging sensor. The local anisotropy of the specimen and tissue morphology can then be observed by investigating these polarization images which are very useful in biomedical imaging [2,3] and material sciences [4,5]. A division of time (DoT) polarimeter is an existing conventional technique adopted in polarization microscopy [6,7]. However, the specimen must be stationary in order to avoid temporal blur. Compared with DoT polarimeter, division of aperture (DoA) and division of focal plane (DoFP) polarimeters allow dynamic measurement [8]; therefore, cellular dynamics and motility can be studied by measuring live cells using these polarimeters as imaging sensor. The spatial resolution is reduced for both polarimeters, but this issue is overcome because of the improvement of the CCD resolution in recent years. A DoA polarimeter is usually large, complex, and requires additional reimaging optics to form multiple polarization images simultaneously. Thus, a DoFP polarimeter which utilizes a compact micropolarizer array is a better choice of an imaging sensor for a polarization microscope. The polarization state of light varies across wavelengths; therefore, most polarization microscopes utilize a monochromatic polarimeter, and the applications determine the operating wavelength of the polarimeter. Near infrared (IR) light, with wavelengths that are just beyond the visible spectrum, experiences less Rayleigh and Mie scattering than visible light, and this characteristic partly contributes to its longer penetration depth. It can penetrate into biological tissues and is useful in bio-imaging applications. Figure 1 shows the absorption spectra of a normal female breast and a normal human forearm [9], and each spectrum has lower absorption at near IR wavelengths. This characteristic allows the polarization imaging of biological tissues using near IR light [10,11]. Compared with other IR wavelengths (1.4 µm −1 mm), near IR light has similar characteristics to visible light, and most solid state devices operating in the visible spectrum have sufficient responsivity at near IR wavelengths. Figure 1 also shows the quantum efficiency of the Truesense KAI-2020 monochromatic CCD (microlens version) utilized in this paper [12]. The responsivity of this CCD is lower in near IR than in visible wavelengths, but it is sufficient for near IR polarization imaging with a proper illumination design.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4358

Fig. 1. The absorption spectra of female breast and human forearm and quantum efficiency of the Truesense KAI-2020 monochromatic CCD (microlens version) [9,12].

In this paper, an array of interference-based liquid crystal polymer (LCP) micropolarizers [13] is fabricated for the construction of a near IR DoFP full-Stokes imaging polarimeter, which is utilized as an imaging sensor of a Mitutoyo microscope. The micropolarizers operating at 760 nm are introduced to extend the capabilities of near IR imaging systems beyond conventional intensity imaging. 760 nm is chosen because of (1) the relatively good sensitivity of the Truesense KAI-2020 sensor, (2) availability of light sources, such as light emitting diodes, that can be utilized as illumination and (3) low absorption of tissue [9]. The 760 nm polarization microscope can be used for sampling the polarization signature of the specimen, and the recorded Stokes images provide important characteristics, such as tissue morphology and crystal composition. The instrument is ideally suitable for transparent anisotropic samples, which have low contrast when viewed with a conventional microscope. Interference-based micropolarizers are utilized here because they generally have higher extinction ratios (ER) and higher transmittance of parallel polarization in the near IR than absorption based LCP micropolarizer based on dichroic dyes [13–15]. In section 2, the design of a 760 nm interference-based micropolarizer is discussed. In section 3, the fabrication processes of the micropolarizer are investigated, and the optical properties of the micropolarizer are presented. The calibration of the polarization sensitive focal plane array is presented in section 4, and the operation of the polarization microscope, along with calibration of different microscope objectives, are presented in section 5. Finally, the unique properties of the polarization microscope are summarized in section 6. 2. Near infrared full-Stokes imaging polarimeter design The optimized design for a full-Stokes polarimeter, utilizing four measurements that form a regular tetrahedron inscribed in the Poincaré sphere [16,17], is applied here in the design of the 760 nm micropolarizer. The interference-based micropolarizer is achieved using the combination of uniform cholesteric LCP (Ch-LCP) circular polarizers, a uniform quarter wave retarder, and a microretarder, which consists of a set of four pixels. Each retarder has 132° retardance with fast axis angles of ± 15.1° and ± 51.7° respectively. The Ch-LCP circular polarizer, which is based on interference, is essential for infrared wavelengths where a good dichroic dye does not exist [13]. At these wavelengths, multi-layer interference-based Ch-LCP thin film stacks with a uniform quarter wave retarder can be used to realize a high ER linear polarizer as a replacement of the absorption-based polarizer in a full-Stokes DoFP polarimeter. Unlike the absorption-based micropolarizer, parallel polarization transmittance of the Ch-LCP film stacks remains high. On the other hand, the Ch-LCP polarizer is utilized instead of other techniques, such as a wire grid polarizer, because the topography of the wire #230499 - $15.00 USD © 2015 OSA

Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4359

grid polarizer impedes the fabrication of the multi-layer structure. Additionally, the Ch-LCP polarizer requires fewer lithographic steps and tools compared to the fabrication of the wire grid polarizer. The optimized micropolarizer design for a 760 nm DoFP full-Stokes polarimeter is shown in Fig. 2. Figure 2(a) shows the multilayer structure of the elliptical interference-based micropolarizer to be installed over the focal plane array (FPA) sensor. The incident light enters through a fused silica substrate on which the multi-layer micropolarizer is formed. The micropolarizer device is comprised of a microretarder, three isolation layers, a uniform 760 nm quarter wave plate, and two uniform 760 nm Ch-LCP polarizers on top of a sensor. Figure 2(b) shows the orientations of the polarization elements. The Ch-LCP films act as right circular polarizers that when combined with a quarter wave plate act like a uniform linear polarizer with the resultant orientation in the vertical direction. The microretarder is comprised of a 2 × 2 pattern of four 132° retarders with fast axis angles of 15.1°, −15.1°, 51.7°, −51.7° with respect to the transmission axis of the uniform resultant linear polarizer as shown in the Fig. 2(b). The thin film stacks result in the elliptical micropolarizer elements shown in Fig. 2(c), where A and B have degree of linear polarization (DOLP) of 0.928 while C and D have DOLP of 0.691. The angle of linear polarization of elliptical micropolarizers A, B, C, and D are 26.7 °, −26.7 °, −73.5 °, and 73.5 ° with respect to the transmission axis of the uniform linear polarizer; the degree of circular polarization (DOCP) are 0.383, −0.383, 0.808, and −0.808. Each micropolarizer element allows a different elliptical polarization state to be transmitted and captured as an intensity signal by its corresponding pixel over the sensor. Figure 2(d) shows this optimized design on the Poincaré sphere, and the four measurements are located at the vertices of a regular tetrahedron.

Fig. 2. (a) The FPA of the polarimeter is comprised of a substrate, a microretarder, a uniform retarder, three isolation layers, and two uniform Ch-LCP polarizers on top of a sensor. (b) Two uniform right circular polarizers, a uniform quarter wave plate, and a pixelated retarder with a retardance of 132° and fast axis angles of ± 15.1° (A, B) and ± 51.7° (C, D) are shown. Dotted lines denote that the micropolarizers are repeated across the sensor array. (c) Each resultant elliptical micropolarizer transmits a different elliptical polarization state and the transmitted intensity is measured by individual pixelated sensor. (d) The four elliptical micropolarizers are utilized to achieve the optimized DoFP full-Stokes polarimeter.

3. Micropolarizer fabrication Ch-LCP is utilized for the fabrication of the uniform circular polarizer layers, and LCP is utilized for the fabrication of the uniform quarter wave plate and the microretarder layer. Here, both the Ch-LCP and the LCP are photo-aligned using linearly photopolymerizable polymers (LPP) from Rolic. A Karl Suss MA6 Mask Aligner with a UV polarizer is used to expose the uniform LPP layer or to pattern the LPP layer. The LPP material is ROP-103 from Rolic. The LCP is RMM141C purchased from EMD Chemicals and is added to Toluene at a 60% w/w ratio for the 132° microretarder fabrication, and at a 51.43% w/w ratio for the uniform quarter wave retarder fabrication. For making the Ch-LCP solution, the LCP material is first added to chloroform at a 20% w/w ratio. Then the chiral dopant, model ZLI-811 from #230499 - $15.00 USD © 2015 OSA

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EMD Chemicals, is added at a 16.3% (w/w) of the concentration of LCP. The substrates are 0.5 mm thick 100 mm diameter double side polished fused silica wafers.

Fig. 3. Fabrication processes of the interference-based elliptical micropolarizer. Note that the dimensions are not drawn to scale.

The 760nm polarimeter utilizes a Truesense KAI-2020 monochromatic CCD (microlens version) with a 1608 × 1208 array and 7.4 μm square pixels. An alignment mark is fabricated by a standard lift-off process with the deposition of chromium. The alignment mark is critical for both defining the alignment orientation of each microretarder element and for accurate dicing of the finished wafers. The positive tone photoresist S1813 and Edwards EB3 e-beam evaporator are utilized, and the size of each micropolarizer element is the same as CCD pixel size which is 7.4 μm square. Figure 3 shows a schematic diagram of the steps involved in the fabrication of the elliptical micropolarizer. The coating of the LPP layer consists of three steps: (1) dispensing Rolic LPP onto the 100 mm wafer, (2) spin coating at 2500 rpm for 1 minute, and (3) soft baking at 170 þC for 6 minutes. Four selective linearly polarized ultraviolet (LPUV) exposures are utilized to register the four different domains for LPP patterning, and each exposure defines the alignment orientation of one quadrant of the macropixel. Each LPUV exposure dose is 100 mJ/cm2 at 365 nm. After patterning the LPP layer, 60% w/w LCP is coated above the LPP layer. The method for coating LCP is (1) spin coating at 2500 rpm for 30 seconds, (2) hard baking at 53 þC for 5 minutes to remove residual solvent, and (3) curing with UV exposure of 1 J/cm2 at 365 nm. After curing, a solid plastic microretarder film is formed. Next, 50 nm SiO2 is sputtered as an isolation layer. Above the isolation layer, a uniform quarter wave retarder is coated using LPP and 51.43% w/w LCP. The second layer of LPP is again spin coated, baked, and exposed to the uniform LPUV. 51.43% w/w LCP is then spin coated above the LPP layer at 2600 rpm. Then 2 minutes of hard baking at 53 þC and UV curing with 1 J/cm2 at 365 nm are applied, and a uniform quarter wave retarder is fabricated above the microretarder layer. After coating the retarder layers, two more uniform Ch-LCP polarizers are coated using LPP and Ch-LCP solution. The processes are exactly the same for the following two layers.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4361

50 nm SiO2 is first sputtered as an isolation layer, and a layer of aligned LPP is prepared. ChLCP is then spin coated above the LPP layer at 3500 rpm, and UV curing with 3 J/cm2 at 365 nm is applied to obtain the Ch-LCP film. The resulting multilayers make up the interferencebased elliptical micropolarizer. The polarization properties of the 760 nm elliptical micropolarizer are measured by a calibrated IR Mueller matrix imaging DoT polarimeter [18]. Figure 4 shows the linear diattenuation, linear diattenuation orientation, and circular diattenuation deduced from the measured Mueller matrices at 760 nm [19,20]. The measured results of elliptical micropolarizers A and C are represented by the blue solid line while those of elliptical micropolarizers B and D are represented by the red dashed lines. The linear diattenuation alternates between 0.2 and 0.7, and the magnitude of circular diattenuation alternates between 0.6 and 0.1. The diattenuation orientation of solid cross-section alternates between 65° and 15°, and dashed cross-section alternates between −10° and −45°. The measured orientation of each fabricated micropolarizer element is shifted from the target design. The differences are attributed mainly to the deviation from the 132° and 90° retardance. Also the magnitude of diattenuation is reduced by the presence of depolarization which comes from scattering in the Ch-LCP layer.

Fig. 4. Horizontal cut lines are shown for linear diattenuation, linear diattenuation orientation, and circular diattenuation taken at 760 nm. The measured results of elliptical micropolarizers A and C are represented by the blue solid line while those of elliptical micropolarizers B and D are represented by the red dashed lines.

After the micropolarizer fabrication, the finished wafer is diced, and an individual die is attached to a mounting frame. A 6-axis stage is used to align the mounted die to the Truesense CCD array. When the die is aligned properly, UV curing epoxy is applied to fix the whole package. The Truesense CCD with the micropolarizer array is then installed in an Imperx ICL-B1620 camera body, and the 760 nm polarimeter is complete.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4362

Fig. 5. (a) A 760 nm collimated light source with a linear polarizer and a quarter wave retarder is utilized for the polarimeter calibration. (b) The DOLP and DOCP are measured as a function of the fast axis orientation of a 90° linear retarder at 760 nm. (c) The measurement errors of DOLP and DOCP are less than 3.5%. (d) The standard deviations of DOLP and DOCP are less than 2%.

4. Polarimeter characterization Before measurement, the 760nm polarimeter must first be calibrated at the target wavelength. The optical setup for the calibration is shown in Fig. 5(a). A 760 nm collimated light source is generated using a 760 nm LED (Marubeni SMBB760D), a 760 nm bandpass filter with 10 nm FWHM, and a 2” collimating lens. The polarization of the collimated light is modified using an IR linear polarizer and a linear retarder of 90° retardance. Six different states of polarized light, 0°, 45°, 90°, 135° linear, and right and left circular polarization illuminate the 760nm polarimeter at normal incidence without the lens. The captured images are then used for computing the calibration matrices. The polarimetric data reduction matrix method is adopted here for the calibration [21]. The performance of the 760 nm polarimeter can be evaluated by uniformly illuminating the 760nm polarimeter with carefully controlled polarized light. The ellipticity of the input polarization is varied by rotating a 90° retarder in front of a 0° linear polarizer, as shown in the optical setup in Fig. 5(a). The average DOLP and DOCP of the measurements are shown in Fig. 5(b) as the retarder rotates from 0° to 180° with 5° increment. The error and standard deviation of the DOLP and DOCP measurements indicate the accuracy and precision. The error in the measurement of the 760nm polarimeter is shown in Fig. 5(c). The maximum DOLP and DOCP errors of the 760nm polarimeter are 3.5%. In Fig. 5(d), the DOLP and DOCP standard deviations, which refer to the uncertainty of the 760nm polarimeter, are shown to be less than 2%. The remaining error and standard deviation are attributed to the defects on the micropolarizer, non-uniformity of the retarder layers, and cross-talk between different pixelated elements. Moreover, the finite bandwidth of the 760 nm bandpass filter results in retardance dispersion and measurement error which cannot be easily removed by calibration. Additionally, the reliability of the calibration as well as DOLP and DOCP measurements are affected by orientation errors in the calibration. In this paper, the rotation of the polarizer and retarder (Fig. 5(a)) are performed manually with an approximate accuracy of about 0.5 °. 5. DoFP polarization microscopy The DoFP polarization microscope is created by removing the standard camera in a Mitutoyo microscope shown in Fig. 6(c) and replacing it with the 760nm polarimeter shown in Fig. 6(b). The KAI-2020 CCD and the fabricated micropolarizer are shown in Fig. 6(a). A one

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4363

inch diameter 760 nm bandpass filter with 10 nm FWHM is installed between the CCD and the microscope adaptor. A Techniquip FOI-150 illuminator with a 150W light bulb and a Dolan-Jenner fiber-based backlight module are utilized as the light source. When measuring the polarization images, a horizontal polarizer is placed in front of the light source, and the polarimetric data reduction matrix [21] is utilized again to derive the Stokes images consisting of four elements S0, S1, S2, and S3. S0 represents the incident intensity of the light; S1 refers to the difference between the horizontal and vertical linear polarizations; S2 denotes the difference between 45° and 135° linear polarizations; S3 expresses the difference between right and left circular polarizations.

Fig. 6. (a) The 760 nm elliptical micropolarizer and the CCD. (b) The camera with the aligned and affixed micropolarizer over the CCD. (c) A Mitutoyo microscope with the 760 nm polarimeter.

On this 760 nm polarization microscope, 2x (NA 0.055), 20x (NA 0.42), 50x (NA 0.55), and 80x (NA 0.5) Mitutoyo Plan Apo microscope objectives are installed. Using the setup similar to Fig. 5 (a), the 760nm polarimeter is calibrated for each microscope objective to minimize the polarization effects from the microscope objectives and the prisms inside the microscope. The DOLP and DOCP accuracy and precision are also evaluated for each microscope objective as shown in Fig. 7. The DOLP and DOCP maximum errors for the 2x, 20x, 50x, and 80x microscope objective are 5.2%, 6.2%, 10.5%, and 6.5%, respectively. The DOLP and DOCP standard deviations are less than 3.6%, 3.7%, 5%, and 5.2%, respectively. The accuracy and precision deteriorate as the NA of microscope objective increases due to both the geometrical and polarization aberrations. Moreover, dust and imperfections inside the microscope and polarization effects from the prisms reduce the signal-to-noise ratio; therefore, the certainty and the sensitivity of the 760nm polarimeter suffer. To enhance the performance, microscope objectives with minimized polarization aberrations at the designed wavelength, 760 nm in this paper, are needed. Also, additional coatings on the prisms to reduce the polarization effects can improve the signal-to-noise ratio.

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Fig. 7. The DOLP and DOCP are also measured as a function of the fast axis orientation of the retarder for 2x, 20x, 50x, and 80x microscope objectives. The measurement error of (a) DOLP and (b) DOCP are less than 5.2%, 6.2%, 10.5%, and 6.5, respectively. The standard deviation of (c) DOLP and (d) DOCP are less than 3.6%, 3.7%, 5%, and 5.2%, respectively.

A polarization image of a Plusiotis batesi beetle is measured under the 2x microscope objective as shown in Fig. 8. A photograph and microscopic image of the beetle are shown in Fig. 8(a), and the corresponding Stokes images are measured under unpolarized illumination as shown in Fig. 8(b). Plusiotis batesi has an external surface consisting of a chitinous cuticle with microscopic structure possessing helicity which results in the circular polarization properties [22,23]. Using the 760 nm full-Stokes polarization microscope, the circular polarized light from the reflection of the beetle’s exoskeleton is easily observed. In the plot of S3, reflection from the Plusiotis batesi shell has a DOCP of up to 0.9.

Fig. 8. (a) The Plusiotis batesi and its microscope image under the 2x microscope objective. (b) The Stokes images of the beetle.

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A polarization image of a Methyl Violet sample which is prepared using crystal violet (C25N3H30Cl) in aqueous and non-aqueous solvent mixture is measured under the 20x microscope objective as shown in Fig. 9. First, the microscopic polarization image is captured using a normal color CCD on the microscope with the sample placed between crossed polarizers as shown in Fig. 9(a), where the variation of colors represents the birefringence of the sample. The crossed polarizers are then removed, and the sample is illuminated using a horizontally polarized light source. The corresponding Stokes images are shown in Fig. 9(b). The local anisotropy of the specimen modulates the input horizontal polarization state; this causes the Stokes vectors to vary across the specimen. Both the linear and circular polarization signatures are successfully retrieved using the 760 nm full-Stokes polarization microscope. Polarization images of tartaric acid and citric acid samples are also measured and shown in Appendix A.

Fig. 9. (a) The polarization image of the methyl violet sample under the 20x microscope objective. (b) The Stokes images of the methyl violet sample.

A polarization image of a Desmidiales, a unicellular freshwater algae, is also captured under the 80x microscope objective as shown in Fig. 10. Similar to the Methyl Violet sample measurement, the microscopic polarization image is captured with the sample placed between crossed polarizers as shown in Fig. 10(a), and the corresponding Stokes images are measured using a horizontally polarized light source as shown in Fig. 10(b). From the plots of S1, S2, S3, and DOLP, the edges of the Desmidiales can be observed because of the birefringence of the cell wall. Since the 760nm full-Stokes polarization microscope can acquire polarization images in a single video frame, living cells can be measured and their cellular dynamics and motility can be studied. An additional polarization image of Desmidiales is shown in Appendix A (see Figs. 11–13).

Fig. 10. (a) The polarization image of Desmidiales under the 80x microscope objective. (b) The Stokes images of Desmidiales.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4366

6. Conclusion In this paper, a polarization microscope using a 760 nm DoFP full-Stokes polarimeter is demonstrated, and the fabrication process of the interference-based LCP elliptical micropolarizer is presented. The elliptical micropolarizer is comprised of uniform Ch-LCP circular polarizers, a uniform quarter wave retarder, and a microretarder. To achieve an optimized polarimeter design, the micropolarizer utilizes a set of four measurements which form a regular tetrahedron inscribed in the Poincaré Sphere. The full-Stokes polarization microscope provides a resolution of 1608 × 1208 × 8-bit at 35 frames/second or 14-bit at 20 frames/second. The DOLP and DOCP error are less than 3.5%, and the precision, defined by the standard deviation, is less than 2%. Future improvement in accuracy and precision can be achieved by improving the uniformity of the retarder layer, decreasing the microretarder element size (to lower pixel-to-pixel cross-talk), reducing polarization effects from the prisms, and utilizing strain-free polarization microscopic objectives. In computing the Stokes images, the polarimetric data reduction matrix method is adopted. Data quality and resolution can be further improved by using interpolation [24,25] and deconvolution techniques [26]. To the best of our knowledge, the 760nm polarimeter used in this microscope is the first DoFP full-Stokes imaging polarimeter operating in the near IR. This work extends the capability of linear LCP micropolarizers to near IR wavelengths by utilizing interferencebased circular micropolarizers. The prototype 760 nm full-Stokes polarization microscope represents a useful application of polarimetry technology. Compared with existing instruments, the polarization microscope is single shot and fast, insusceptible to vibration and sample motion, compact and sensitive to all polarization states, including linear, circular and elliptical. Rapid measurements of degree of polarization, degree of linear polarization, and degree of circular polarization can provide valuable qualitative and quantitative information in biological imaging and material sciences. Future research can include extension to broadband operation utilizing multi-layer achromat optical filters [13,27]. Appendix A: Microscopic polarization Images

Fig. 11. (a) The polarization image of the tartaric acid sample under the 20x microscope objective. (b) The Stokes images of the tartaric acid sample.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4367

Fig. 12. (a) The polarization image of the citric acid sample under the 20x microscope objective. (b) The Stokes images of the citric acid sample.

Fig. 13. (a) The polarization image of Desmidiales under the 80x microscope objective. (b) The Stokes images of Desmidiales.

Acknowledgments This work is funded by University of Arizona Proof of Concept Award, the Arizona Technology Research Infrastructure Fund (TRIF), and the Air Force Research Laboratories STTR grant number FA8651-13-M-0085 in collaboration with the Spectral Imaging Laboratory (Pasadena, CA). The authors thank Prof. Russell Chipman’s research groups for allowing us to utilize their equipment and Prof. Arthur Gmitro for the Desmidiales samples.

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Received 12 Dec 2014; revised 4 Feb 2015; accepted 5 Feb 2015; published 11 Feb 2015 23 Feb 2015 | Vol. 23, No. 4 | DOI:10.1364/OE.23.004357 | OPTICS EXPRESS 4368

Polarization microscope using a near infrared full-Stokes imaging polarimeter.

This paper presents a polarization microscope using an infrared (IR) full-Stokes imaging polarimeter. The IR polarimeter utilizes an optimized interfe...
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