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1.IntroductionMicroarrays are widely used in genomic, proteomic, and diagnostic applications.1 A microarray consists of specific biomolecules (oligonucleotide sequences, polymerase chain reaction products, proteins, etc.) immobilized as spots on a substrate surface. The microarray is then reacted with a sample containing analytes that specifically bind to immobilized biomolecules. Typically, the analytes are labeled with fluorescent dye(s) and the intensity of the bound fluorescent dye is read on a fluorescent microarray reader. The fluorescence intensity is related to the concentration of analytes bound. Fluorescence-based microarray reading methods offer high sensitivity. The ability to use multiple fluorescent dyes that fluoresce at different wavelengths offers the attractive choice, where control and samples are to be tested in a single measurement. One of the drawbacks in using fluorescent labels arises from the additional steps that are involved in the attachment of labels to analytes. Other drawbacks are the requirement for additional steps of purification of the fluorescent labeled analytes and the photostability of fluorescent dyes. Finally, the attachment of a label to the analyte can alter its binding characteristics and may not provide a realistic picture of its biological activity. Therefore, alternative label-less methods to read microarrays are in development.2 This paper describes an approach to microarray readout that does not require any labels. Utilizing evanescent imaging ellipsometry, this method offers a highly sensitive alternative to fluorescent microarray readers. The following sections describe the theoretical principles of the imaging ellipsometric method, the construction of the optical detection unit, and the fabrication of microarrays for testing and the characterization of microarray readers. 2.Ellipsometric Microarray Reader Optical SystemFigure 1 shows a schematic diagram of the imaging ellipsometric microarray reader that is based on a patent by Kempen.3 The light source is a collimated green 520-nm LED. A polarizer and a quarter-wave linear retarder in rotary stages generate polarized light with adjustable orientation and ellipticity. This light enters a right-angle prism at normal incidence. The microarray is fabricated on a poly-l-lysine-coated BK7 glass slide. The microarray is mounted horizontally and the bottom surface of the microarray is affixed to the hypotenuse of the prism with index-matching fluid, as shown in Fig. 2 . In this configuration, the incident angle on the microarray, , is larger than the critical angle of . The polarization state change at the microarray interface is primarily due to the retardance associated with total internal reflection (TIR) at a dielectric interface with additional modification due to interaction with the microarray spots. The total internally reflected light then exits the prism at normal incidence. The polarizer and retarder’s orientations are adjusted such that the light reflected from the BK7 interface is linearly polarized at . An analyzer (linear polarizer) is oriented at a 45-deg orientation to extinguish the TIR beam. The binding of analytes to the microarray spots, cause thickness changes and changes in complex refractive index , across the surface of the microarray sample. The vector is position on the microarray. These changes cause an additional alteration to the polarization state of the beam reflected from the TIR interface, inducing a small leakage of light at the polarizer. Two folding mirrors return the beam to a horizontal axis. The light leaked through the analyzer constitutes the signal. A telephoto camera lens set at images the microarray onto a scientific grade 10-bit CCD camera (Optronics, MagnaFire). A photograph of the imaging ellipsometric microarray reader prototype is shown in Fig. 3 . This beam has a limiting resolution of . The CCD pixel size also limits the resolution to about . Despite the tilted image plane, the images were well resolved over a 1-cm field of view. 3.Principle of OperationThe ellipsometric microarray reader measures the change in the polarization state of the light reflected from the microarray surface to determine the concentration of bound analytes to microarray spots. This change is modeled using the equations of optical thin films and ellipsometry.4, 5, 6 At the microarray interface, the light beam undergoes TIR from the hypotenuse surface of a glass prism in contact with a microarray sample.7, 8 The reflection causes separate changes in the amplitudes and phases of the and components of the incident light field, which are characterized by two complex amplitude reflection coefficients and . For a BK-7 glass slide without microarray spots, and are the Fresnel reflection coefficients. For a glass slide containing layers of biomolecules, and are calculated by treating the biomolecules as a thin film coating. The retardance is defined as the relative change in phase between the and components of the light field and is given by The retardance and the changes in amplitude between and components, , are characterized in ellipsometry byThe change in the amplitudes of and , which characterize the reflection amplitude differences for different polarization states is also characterized by the parameter diattenuation defined asa measure of partial polarization. Diattenuation varies from 0, when all polarization states have equal reflectance (such as TIR), to 1 for an ideal polarizer. When a light field undergoes TIR in nonabsorbing materials, only retardance occurs since and . In this configuration, changes to arise from both variations in thickness and refractive index. When the biomolecules have absorption, a nonzero diattenuation occurs.To obtain a linearly polarized beam at following TIR, the polarizer and the quarter-wave plate azimuthal are selected such that the incident amplitudes of the and components are equal with a difference in phase opposite to the TIR retardance. As shown later, this beam should have Jones vector Then the internally reflected beam has the Jones vectorThe retardance and diattenuation of the interface are represented by Jones matrix ,The reflected beam is almost completely blocked by an analyzer, a linear polarizer oriented at with a Jones matrixA lens relays the light to a CCD array. For a bare glass slide, the reflected beam is completely extinguished by the analyzer.The retardance and diattenuation introduced by reflection from the spots on microarray surface cause the totally internally reflected beam to become elliptically rather than linearly polarized with Jones vectorThe diattenuation and retardance of the microarray spots prevents the light from being completely extinguished by the 135-deg analyzer because the Jones vector transmitted through the 135-deg polarizer isThus, retardance changes cause elliptically polarized light. Diattenuation causes the axis of the polarization to rotate from . These changes depend on , and . The intensity of the microarray image becomeswhere the spatial dependence is suppressed.For film thicknesses much smaller than the optical wavelength, the retardance change is small, leading to the approximate result Similarly, since and , the diattenuation is small and can be approximated asApplying these approximations, the image intensity becomesIn summary, spatial variations in and that occur due to the biomolecules on the microarray surface result in a nonuniform intensity distribution described by Eq. 14, which is measured by the ellipsometric microarray reader’s CCD. Since the variations of and in the image are associated with the spatial variations , , and of the sample film, the image intensity provides information about these optical parameters of the sample film.The Poincaré sphere in Fig. 4 helps explain the evolution of the light state through the polarizer, quarter wave retarder, TIR, and the small influence of film diattenuation and retardance. The objective of the setup is to end at , 45-deg linearly polarized light. Light exits the polarizer at a position and is rotated to by the quarter-wave retarder oriented at . TIR from the bare surface, a retarder at , brings the state to , linearly polarized at , which will be extinguished by the final oriented analyzer. The polarizer azimuth is set at , where is the retardance introduced by TIR. Any change in retardance introduced by the microarray causes the polarization state to move along the longitude of the Poincaré sphere. Diattenuation caused by the microarray moves the polarization state along the equator. Both cause leakage at the final analyzer. Additionally, an adjustment of the initial polarizer azimuth can differentiate between the diattenuation and retardance. For our prototype ellipsometric reader, the BK7 prism refractive index is , the angle of incidence is , and the microarray surface is immersed in air . A typical biomolecular layer has an index of refraction in a range and a thickness of approximately (Refs. 2, 9, 10). For the nucleotide sequence of about 30 nucleic acids we estimate the index of refraction to be . A thin film calculation6 using these parameters demonstrates that a biomolecular layer introduces a retardance change, , from the bare interface of which is readily measured by this technique.The sensitivity to changes in refractive index and thickness is found by differentiating with respect to index and thicknessThe refractive index sensitivity and the layer thickness sensitivity establish the requirements for determining small thickness and index variation. These requirements include the purity of the incident polarization state, the extinction coefficient of the polarizers, and any intrinsic depolarization caused by the biomolecule spots.The sensitivity can be improved by optimizing the prism refractive index and incident angle. Figure 5 shows the refractive index sensitivity and the layer thickness sensitivity for the typical biomolecular layer ( and ) as functions of substrate refractive index and angle of incidence . An examination of Fig. 5 indicates the following:
4.Measurements and Results4.1.Fabrication of MicroarraysThree OMM16K mouse complementary DNA (cDNA) arrays are used to test the imaging ellipsometric microarray reader. The microarrays contain 70-mer oligonucleotides deposited as spots printed on poly-l-lysine-coated slides in 32 subarrays of 24 columns and 23 rows each for a total of 17,664 spots using Telechem Stealth SMP3 pins in 32 pin configuration. The spots are separated by a center- to-center distance of . The array size is . Cy3-labeled arrays were tested to make a direct comparison of imaging ellipsometer measurements to microarray scanners that utilize Cy3 fluorescent labels. The array description and gene list can be found in Ref. 11. Figure 6 outlines the steps to hybridize cDNA to microarray. Total RNA was prepared using Qiagen RNeasy Midi Kit. The messenger RNA (mRNA) was prepared from total RNA using Oligotex columns from Qiagen. of total RNA was used for each labeling reaction. Prior to labeling, total RNA was concentrated to about using ethanol/isopropanol precipitation (1 vol. RNA+2 vol. ethanol:isopropanol 1:1). A 10-min precipitation at room temperature was followed by a 10-min spin at in a microfuge to recover the nucleic acids. The RNA was denatured and primers were annealed by incubating of resuspended RNA, of oligo dT and random hexamers for at . The first strand synthesis and labeling was performed by adding first strand buffer, , each dACG, dTTP, aa-dUTP, RNAse to of denatured RNA. Do microliters of Superscript-RT (Gibco) was added to this mixture and incubated at for . This was followed by heating the mixture to for 2 to . The RNA was removed by adding EDTA and and heating for at 60 to . Then HCl and Tris pH 7.5 was added to neutralize . The cDNA probe was cleaned using CyScribe GFX purification kit (Amersham Biosciences, catalog #27-9606-01). Cy3-NHS was disordered in DMSO three concentration of . Then of this dye was added to each cDNA probe and incubated at room temperature for 60 to . After incubation, the reaction was halted by adding ethanolamine at room temperature for . The labeled probe was purified by Cyscribe GFX purification kit. The purified dye labeled probe was denatured by mixing probe with , poly dA and hybridization buffer. The mixture was heated to for . Next of prehybridization buffer was added on the microarray slide and the microarray was covered with a coverslip and placed in the hybridization chamber for at . The prehybridization buffer was rinsed with water, 70% ethanol, and 100% ethanol and air dried. These microarrays were used for hybridization and testing. The of dye-labeled probes are added on top of the prehybridized microarray slides. The slide was covered with a cover slip and hybridized for in a hybridization chamber. These hybridize microarrays were tested by fluorescent microarray scanner and imaging ellipsometer. 4.2.Measurement ProcedureThe hypotenuse surface of the prism is cleaned using standard optical cleaning methods. A drop of refractive index matching fluid is placed on one edge of the hypotenuse surface and the rear surface of the glass slide is slowly attached to the hypotenuse of the prism allowing the index matching fluid to spread between the prism and the glass slide surfaces, sealing the gap. Caution is taken to avoid air bubbles trapped in the imaging area, which cause bright spots on the ellipsometric image. An image is formed of the microarray on the CCD and the polarization elements in the ellipsometer are adjusted to achieve the best null possible in the area surrounding the microarray spots. The lens is stopped down to to increase the depth of focus to accommodate the tilted object plane. The Cy3-labeled probes are used to compare the results from imaging ellipsometry experiments to those from a fluorescence microarray reader (Axon Instruments). The fluorescent measurement of the Cy3-hybridized microarray is shown in Fig. 7a . The spots are clearly visible and the measurements show bright spots with a clear dark background. Figure 7b shows the ellipsometric image of a similar microarray using the prototype ellipsometric microarray reader. Note that the sensitivity analysis already given demonstrated that the change in retardance caused by the microarray spots is very small. Therefore, to achieve the necessary sensitivity, the image exposure time was set to about to increase the retardance sensitivity of the instrument resulting in the camera the dynamic range being significantly smaller than the total contrast ratio of the ellipsometer. The extinction ratio of the ellipsometer was measured to be about 3000:1, and we believe this is limited by retarder uniformity and scattering within the prism. 4.3.Calibration ProcedureThe microarray reader is calibrated using a high-precision imaging Mueller matrix polarimeter (IMMP), located at the Polarization Laboratory of the Optical Science Center, University of Arizona. Figure 8 shows a schematic diagram of the IMMP system. A detailed description of this system can be found in the literature. 7, 12, 13, 14 Mueller matrix images are then reduced to retardance and diattenuation maps using a polar decomposition algorithm.15 Calibration is performed by first using the ellipsometer to acquire an image of a microarray sample. A cross section of the image is extracted and the average of the intensity variations induced by the microarray spots is determined. Next the IMMP system is used to measure the retardance caused by TIR in the glass prism and the retardance change induced by the microarray spots. The intensity variation of the ellipsometer image is proportional to the square of the retardance deviation, that is, . From the IMMP retardance image, the constant in this equation is determined. 4.3.1.Estimating the average intensity variations induced by the microarray pixelsThe microarray image is first highpass filtered in the spatial frequency domain to remove the gradual variation in the background intensity. Figure 9 is a cross section of the image along a row of spots normalized by the digital range of the detector. The average intensity variation induced by the microarray spots is measured to be 8% of digital range of the CCD detector. 4.3.2.Estimating the retardance deviation induced by the microarray pixelsTo determine the relationship between the image intensity and microarray spot thickness, the retardance change and the diattenuation of the microarray sample and prism are independently measured by the IMMP system. The resolution of the IMMP enables the retardance and diattenuation variations to be measured to an accuracy of better than 1%. The light paths through the prism in the IMMP measurement are set to match the microarray imaging ellipsometer configuration: The wavelength is , and the angle of incidence is . The retardance introduced by the microarray is the dominant polarization effect contributing to the image intensity. The diattenuation introduced by the microarray sample is small , which lies in the noise of the IMMP. Figure 10 shows a retardance image of the microarray measured by the IMMP system. From the and cross sections, the mean retardance deviation introduced by a microarray spot, relative to the background (local maximum to local minimum) is measured to be Assuming thickness variation at constant refractive index, then is related to the sample’s thickness by Eq. 17, We estimate the mean thickness change of the microarray spots asThis thickness change is independently confirmed by a Wyko-NT2000 phase shifting interferometer measurement shown in Fig. 11 confirming that the spots are about thick.4.4.System CalibrationThe retardance calibration is based on finding the constant in the equation Usingyieldswhere is the fraction of total power incident at the prism reaching the CCD.According to the Drude approximation,16 the retardance change is proportional to the thickness variation of the extremely thin film. Thus, the calibration equation for thickness variation is withyieldswhere is the value in nanometers, and has units of .Using Equation 16, a calibration relationship between refractive index and image intensity is given by where is the change in refractive index from a value of 1.44. Using the value from Eq. 16, and from Eq. 22 yields4.5.Ellipsometer SensitivityThe thickness sensitivity of the ellipsometer system can be estimated by rearranging Eq. 23 and differentiating with respect to intensity. The term is the uncertainty in the measured intensity, caused by noise in the camera which is estimated usingwhere is the number of incident photons, QE is the quantum efficiency of the camera (42% for our camera). Dark current is (over the exposure time), and the read noise is . The term in the denominator represents the effects of shot-noise on the camera intensity reading. From Fig. 9, the mean intensity signal is 16% of the dynamic range of the camera yielding , which results in a , or . Using Eq. 28 the thickness sensitivity of the ellipsometer is . Using a similar analysis, the index sensitivity of the ellipsometer is .5.AnalysisThe primary goal is to measure the binding of analytes to the biomolecules immobilized on the microarray. The change in intensity of the microarray spots shown in Fig. 7b demonstrate that the evanescent imaging ellipsometric microarray reader is capable of measuring a thickness change of less than and a refractive index change of smaller than 0.0025. Nevertheless, the comparison with the measurement in Fig. 7a shows that the sensitivity to molecular binding is significantly lower than the sensitivity obtained using fluorescence measurements. The difference in sensitivity is due to the fact that the binding of analytes only causes a thickness change on the order of . The sensitivity analysis shown in Fig. 5 demonstrates that the sensitivity of the ellipsometry system can be improved substantially by using a prism made from high-index glass (such as SF11 with an index of ). However, high-index glass prisms are complicated due to difficulties with high-refractive-index-matching fluids. 6.ConclusionsThe development of a label-less imaging ellipsometric microarray reader based on imaging ellipsometry was described. A prototype imaging ellipsometric system was constructed and oligonucleotide microarray samples were tested. The microarray reader is calibrated against an imaging polarimeter and a commercial interferometer. The system’s thickness sensitivity is and the sensitivity to refractive index changes is 0.0024. AcknowledgmentsThe authors thank the National Aeronautics and Space Administration (NASA) for financial support of this work through Contract No. NNM04AA24C. ReferencesS. Venkatasubbarao,
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