Cross-polarization Scheme Research Articles

Carbon-detected deuterium solid-state NMR rotating frame relaxation measurements for protein methyl groups under magic angle spinning

Deuterium rotating frame solid-state NMR relaxation measurements (2H R1ρ) are important tools in quantitative studies of molecular dynamics. We demonstrate how 2H to 13C cross-polarization (CP) approaches under 10–40 kHz magic angle spinning rates can be combined with the 2H R1ρ blocks to allow for extension of deuterium rotating frame relaxation studies to methyl groups in biomolecules. This extension permits detection on the 13C nuclei and, hence, for the achievement of site-specific resolution. The measurements are demonstrated using a nine-residue low complexity peptide with the sequence GGKGMGFGL, in which a single selective −13CD3 label is placed at the methionine residue. Carbon-detected measurements are compared with the deuterium direct-detection results, which allows for fine-tuning of experimental approaches. In particular, we show how the adiabatic respiration CP scheme and the double adiabatic sweep on the 2H and 13C channels can be combined with the 2H R1ρ relaxation rates measurement. Off-resonance 2H R1ρ measurements are investigated in addition to the on-resonance condition, as they extent the range of effective spin-locking field.

Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR.

INEPT-based experiments are widely used for 1 H→15 N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hwater HN exchange process to boost the 1 H→15 N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hwater and HN protons by a strong 1 H RF field, while fulfilling the γH B1,H =γN B1,N Hartmann-Hahn matching condition. Given the low value of γN /γH , however, these demands are often incompatible-particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.

Effects of illuminance intensity on the green channel of remote photoplethysmography (rPPG) signals.

Point-of-care remote photoplethysmography (rPPG) devices that utilize low-cost RGB cameras have drawn considerable attention due to their convenience in contactless and non-invasive vital signs monitoring. In rPPG, sufficient lighting conditions are essential for obtaining accurate diagnostics by observing the complete signal morphology. The effects of illuminance intensity and light source settings play a significant role in rPPG assessment quality, and it was previously observed that different lighting schemes result in different signal quality and morphology. This study presents a quantitative empirical analysis where the quality and morphology of rPPG signals were assessed under different light settings. Participants' faces were exposed to the white LED spotlight, first when the sources were installed directly behind the video camera, and then when the sources were installed in a cross-polarized scheme. Hence, the effect of specular reflectance on rPPG signals could be observed in an increasing projection. The signal qualities were analyzed in each intensity level using a signal-to-noise (SNR) ratio metric. In 3 of 7 participants, placing the video camera on the same level as the light source led to signal quality loss of up to 3 dB for the range 30-60 Lux. In addition, two fundamental morphological features were analyzed, and the derivative-related feature was found to be increasing with illuminance intensity in 6 of 7 participants.

On the use of cross polarization in solid-state NMR: 1H spin-lock versus adiabatic demagnetization in the rotating frame

Cross polarization (CP) is a widely used solid-state nuclear magnetic resonance (NMR) technique for enhancing the polarization of dilute S spins from much larger polarization of abundant I spins such as 1H. To achieve such a polarization transfer, the I spin should either be spin-locked or be converted to the dipolar ordered state through adiabatic demagnetization in the rotating frame. In this work, we analyze the spin dynamics of the Hartmann-Hahn CP (HHCP) utilizing the 1H spin-locking, and the dipolar-order CP (DOCP) having the 1H adiabatic demagnetization. We further propose an adiabatic demagnetization CP (ADCP) where a constant radio-frequency pulse is applied on the S spin while 1H is adiabatically demagnetized. Our analyses indicate that ADCP utilizes the adiabatic passage to effectively achieve the polarization transfer from the 1H to S spins. In addition, the dipolar ordered state generated during the 1H demagnetization process could also be converted into the observable S polarization through DOCP, further enhancing the polarized signals. It is shown by both static and magic-angle-spinning (MAS) NMR experiments that ADCP has dramatically broadened the CP matching condition over the other CP schemes. Various samples have been used to demonstrate the polarization transfer efficiency of this newly proposed ADCP scheme.

Sensitivity Enhancement by Progressive Saturation of the Proton Reservoir: A Solid-State NMR Analogue of Chemical Exchange Saturation Transfer.

Chemical exchange saturation transfer (CEST) enhances solution-state NMR signals of labile and otherwise invisible chemical sites, by indirectly detecting their signatures as a highly magnified saturation of an abundant resonance—for instance, the 1H resonance of water. Stimulated by this sensitivity magnification, this study presents PROgressive Saturation of the Proton Reservoir (PROSPR), a method for enhancing the NMR sensitivity of dilute heteronuclei in static solids. PROSPR aims at using these heteronuclei to progressively deplete the abundant 1H polarization found in most organic and several inorganic solids, and implements this 1H signal depletion in a manner that reflects the spectral intensities of the heteronuclei as a function of their chemical shifts or quadrupolar offsets. To achieve this, PROSPR uses a looped cross-polarization scheme that repeatedly depletes 1H–1H local dipolar order and then relays this saturation throughout the full 1H reservoir via spin-diffusion processes that act as analogues of chemical exchanges in the CEST experiment. Repeating this cross-polarization/spin-diffusion procedure multiple times results in an effective magnification of each heteronucleus’s response that, when repeated in a frequency-stepped fashion, indirectly maps their NMR spectrum as sizable attenuations of the abundant 1H NMR signal. Experimental PROSPR examples demonstrate that, in this fashion, faithful wideline NMR spectra can be obtained. These 1H-detected heteronuclear NMR spectra can have their sensitivity enhanced by orders of magnitude in comparison to optimized direct-detect experiments targeting unreceptive nuclei at low natural abundance, using modest hardware requirements and conventional NMR equipment at room temperature.

Protonation tuned dipolar order mediated 1H→13C cross-polarization for dissolution-dynamic nuclear polarization experiments

A strategy of dipolar order mediated nuclear spin polarization transfer has recently been combined with dissolution-dynamic nuclear polarization (dDNP) and improved by employing optimized shaped radiofrequency pulses and suitable molecular modifications. In the context of dDNP experiments, this offers a promising means of transferring polarization from high-gamma 1H spins to insensitive 13C spins with lower peak power and lower energy compared with state-of-the-art cross-polarization schemes. The role of local molecular groups and the glassing matrix protonation level are both postulated to play a key role in the polarization transfer pathway via an intermediary reservoir of dipolar spin order. To gain appreciation of the mechanisms involved in the dipolar order mediated polarization transfer under dDNP conditions, we investigate herein the influence of the pivotal characteristics of the sample makeup: (i) revising the protonation level for the constituents of the DNP glass; and (ii) utilizing deuterated molecular derivatives. Experimental demonstrations are presented for the case of [1–13C]sodium acetate. We find that the proton sample molarity has a large impact on both the optimal parameters and the performance of the dipolar order mediated cross-polarization sequence, with the 13C signal build-up time drastically shortened in the case of high solvent protonation levels. In the case of a deuterated molecular derivative, we observe that the nearby 2H substituted methyl group is deleterious to the 1H→13C transfer phenomenon (particularly at low levels of sample protonation). Overall, increased solvent protonation makes the dipolar order governed polarization transfer significantly faster and more efficient. This study sheds light on the influential sample formulation traits which govern the dipolar order-controlled transfer of polarization and indicates that the polarization transfer efficiencies of deuterated molecules can be boosted and reach high performances simply by adequate solvent protonation.

Decoherence optimized tilted-angle cross polarization: A novel concept for sensitivity-enhanced solid-state NMR using ultra-fast magic angle spinning

Ultra-fast magic-angle spinning (UFMAS) at a MAS rate (ωR/2π) of 60 kHz or higher has dramatically improved the resolution and sensitivity of solid-state NMR (SSNMR). However, limited polarization transfer efficiency using cross-polarization (CP) between 1H and rare spins such as 13C still restricts the sensitivity and multi-dimensional applications of SSNMR using UFMAS. We propose a novel approach, which we call decoherence-optimized tilted-angle CP (DOTA CP), to improve CP efficiency with prolonged lifetime of 1H coherence in the spin-locked condition and efficient band-selective polarization transfer by incorporating off-resonance irradiation to 1H spins. 13C CP-MAS at ωR/2π of 70–90 kHz suggested that DOTA CP notably outperformed traditional adiabatic CP, a de-facto-standard CP scheme over the past decade, in sensitivity for the aliphatic-region spectra of 13C-labeled GB1 protein and N-formyl-Met-Leu-Phe samples by up to 1.4- and 1.2-fold, respectively. 1H-detected 2D 1H/13C SSNMR for the GB1 sample indicated the effectiveness of this approach in various multidimensional applications.

Dipolar order mediated <sup>1</sup>H → <sup>13</sup>C cross-polarization for dissolution-dynamic nuclear polarization

Abstract. Magnetic resonance imaging and spectroscopy often suffer from a low intrinsic sensitivity, which can in some cases be circumvented by the use of hyperpolarization techniques. Dissolution-dynamic nuclear polarization offers a way of hyperpolarizing 13C spins in small molecules, enhancing their sensitivity by up to 4 orders of magnitude. This is usually performed by direct 13C polarization, which is straightforward but often takes more than an hour. Alternatively, indirect 1H polarization followed by 1H→13C polarization transfer can be implemented, which is more efficient and faster but is technically very challenging and hardly implemented in practice. Here we propose to remove the main roadblocks of the 1H→13C polarization transfer process by using alternative schemes with the following: (i) less rf (radiofrequency) power; (ii) less overall rf energy; (iii) simple rf-pulse shapes; and (iv) no synchronized 1H and 13C rf irradiation. An experimental demonstration of such a simple 1H→13C polarization transfer technique is presented for the case of [1-13C]sodium acetate, and is compared with the most sophisticated cross-polarization schemes. A polarization transfer efficiency of ∼0.43 with respect to cross-polarization was realized, which resulted in a 13C polarization of ∼8.7 % after ∼10 min of microwave irradiation and a single polarization transfer step.

Excited states of neutral and charged excitons in single strongly asymmetric InP-based nanostructures emitting in the telecom C band

We investigate strongly asymmetric self-assembled nanostructures with one of dimensions reaching hundreds of nanometers. Close to the nanowire-like type of confinement, such objects are sometimes assigned as one-dimensional in nature. Here, we directly observe the spectrum of exciton excited states corresponding to longitudinal quantization. This is based on probing the optical transitions via polarization-resolved microphotoluminescence excitation ($\mu$PLE) measurement performed on single nanostructures combined with theoretical calculation of neutral and charged exciton optical properties. We successfully probe absorption-like spectra for individual bright states forming the exciton ground-state fine structure, as well as for the negatively charged exciton. Confronting the calculated spectrum of excitonic absorption with $\mu$PLE traces, we identify optical transitions involving states that contain carriers at various excited levels related to the longest dimension. Based on cross-polarized excitation-detection scheme, we show very well conserved spin configuration during orbital relaxation of the exciton from a number of excited states comparable to the quasi-resonant pumping via the optical phonon, and no polarization memory for the trion, as theoretically expected.

A Digital Shade-Matching Device for Dental Color Determination Using the Support Vector Machine Algorithm.

In this study, we developed a digital shade-matching device for dental color determination using the support vector machine (SVM) algorithm. Shade-matching was performed using shade tabs. For the hardware, the typically used intraoral camera was modified to apply the cross-polarization scheme and block the light from outside, which can lead to shade-matching errors. For reliable experiments, a precise robot arm with ±0.1 mm position repeatability and a specially designed jig to fix the position of the VITA 3D-master (3D) shade tabs were used. For consistent color performance, color calibration was performed with five standard colors having color values as the mean color values of the five shade tabs of the 3D. By using the SVM algorithm, hyperplanes and support vectors for 3D shade tabs were obtained with a database organized using five developed devices. Subsequently, shade matching was performed by measuring 3D shade tabs, as opposed to real teeth, with three additional devices. On average, more than 90% matching accuracy and a less than 1% failure rate were achieved with all devices for 10 measurements. In addition, we compared the classification algorithm with other classification algorithms, such as logistic regression, random forest, and k-nearest neighbors, using the leave-pair-out cross-validation method to verify the classification performance of the SVM algorithm. Our proposed scheme can be an optimum solution for the quantitative measurement of tooth color with high accuracy.

Design of 1-Bit Coding Engineered Reflectors for EM-Wave Shaping and RCS Modifications

In this paper, the engineered reflectors are designed and characterized for cross polarization rotation, RCS modification, and EM-wave shaping at W-band. The multiple plasmon resonances, cross-polarization rotation, reflection phase cancellation, and coding sequence principles are combined together to design the presented reflectors. First, an anisotropic unit cell consisting of a two E-shaped metallic resonators on the top side of a PEC-backed dielectric substrate is precisely designed and optimized. The unit cell operates in a linear cross-polarization scheme from about 86 GHz to 94 GHz and has multiple plasmon resonances at 86.5 GHz, 89.2 GHz, 92.2 GHz, and 93.2 GHz with 100% cross-polarization conversion efficiency at these frequencies. Then this unit cell and its mirrored unit cell are used to compose a number of 1-bit coding reflective engineered reflectors to generate the “1” and “0” elements of the coding sequence required for EM-wave shaping. Four engineered reflectors of various coding sequences are designed to shape the backscattered energy to achieve one lobe, two lobes, three lobes, and four lobes. Furthermore, the low-scattering diffuse reflection pattern is also achieved under both normal and oblique incidence by using a random distribution (random coding sequence) of the unit cells across the engineered reflector aperture. Both 3D full wave simulations and measurement results verify the capability of the presented surfaces in shaping the backscattered EM-wave.

Transient NOE enhancement in solid-state MAS NMR of mobile systems

It has been known that the heteronuclear cross-relaxation affects the dilute S spin magnetization along the longitudinal direction, causing an overshoot phenomenon for those mobile systems in spin-lattice relaxation rate measurements. Here, we analyze the Solomon equations for an I-S system and derive the transient cross relaxation effect as to when an overshoot phenomenon would take place and what the maximum enhancement could be at the time of the overshoot. In order to utilize such a transient nuclear Overhauser effect (NOE), we first time apply it to dynamic solid samples by inverting the 1H magnetization prior to the excitation of the S spin. It is found that the overshoot depends on the ratio of the I and S spin-lattice relaxation rates, i.e. RSS/RII. When RSS/RII≫1, the maximum enhancement factor for transient NOE could be larger than that obtained in steady-state NOE experiments. Furthermore, transient NOE appears to be more efficient in terms of sensitivity enhancement of dilute spins in solid-state NMR of mobile systems than the traditional cross polarization scheme whose efficiency is greatly compromised by molecular mobility. A sample of natural abundance l-isoleucine amino acid, in which the spin-lattice relaxation rates for the four methyl carbons are different, has been used to demonstrate sensitivity enhancement factors under various experimental schemes.

Cross-Polarized Surface-Enhanced Infrared Spectroscopy by Fano-Resonant Asymmetric Metamaterials

Plasmonic metamaterials have overcome fundamental limitations in conventional optics by their capability to engineer material resonances and dispersions at will, holding great promise for sensing applications. Recent demonstrations of metamaterial sensors, however, have mainly relied on their resonant nature for strong optical interactions with molecules, but few examples fully exploit their functionality to manipulate the polarization of light. Here, we present cross-polarized surface-enhanced infrared absorption (SEIRA) by the Fano-resonant asymmetric metamaterial allowing for strong background suppression as well as significant field enhancement. The metamaterial is designed to exhibit the controlled Fano resonance with the cross-polarization conversion property at 1730 cm−1, which spectrally overlaps with the C=O vibrational mode. In the cross-polarized SEIRA measurement, the C=O mode of poly(methyl methacrylate) molecules is clearly observed as a distinct dip within a Fano-resonant transmission peak of the metamaterial. The vibrational signal contrast is then improved based on the cross-polarized detection scheme where only the light interacting with the metamaterial-molecular coupled system is detected by totally eliminating the unwanted background light. Our metamaterial approach achieves the zeptomole sensitivity with a large signal-to-noise ratio in the far-field measurement, paving the way toward the realization of ultrasensitive IR inspection technologies.

High-efficiency low-crosstalk dielectric metasurfaces of mid-wave infrared focal plane arrays

High-resolution compact-size focal plane arrays (FPAs) suffer the fundamental geometrical tradeoff between the optical resolution (pixel size miniaturization) and the optical crosstalk (spillover of neighboring pixel focusing). For FPAs, our previously reported metallic metasurfaces reached an unprecedented level of crosstalk suppression. However, practical utilization of these metallic microlens arrays has proved to be intrinsically limited due to the low device efficiency (of the order of 0.10) resulting from the fundamental absorption losses of metals and their cross-polarization scheme. Exceeding this limit, here we show highly efficient microlens designs enabled by dielectric metasurfaces for mid-wave infrared (MWIR) operation. These dielectric MWIR FPAs allow for a substantially high device efficiency over 0.80 without compromising the optical crosstalk performance. Systematically studying dielectric nanoantennas of silicon nanodisks that do not dictate the cross-polarization scheme using full-wave solutions, we found that the optical crosstalk is suppressed to low levels ≤ 3.0% while sustaining the high efficiency. A figure-of-merit (FoM) defined for the device performance as the focusing efficiency per optical crosstalk times the f-number achieves 84, which is superior to all other types of MWIR FPAs reported to date, all falling below a maximum FoM of 70. These findings indicate that the proposed approach can pave the way for the practical usage of metasurface microlens arrays in MWIR.

An Ultra-wideband and Polarization-independent Metasurface for RCS Reduction.

In this paper, an ultra-wideband and polarization-independent metasurface for radar cross section (RCS) reduction is proposed. The unit cell of the metasurface operates in a linear cross-polarization scheme in a broad band. The phase and amplitude of cross-polarized reflection can be separately controlled by its geometry and rotation angle. Based on the diffuse reflection theory, a 3-bit coding metasurface is designed to reduce the RCS in an ultra-wide band. The wideband property of the metasurface benefits from the wideband cross polarization conversion and flexible phase modulation. In addition, the polarization-independent feature of the metasurface is achieved by tailoring the rotation angle of each element. Both the simulated and measured results demonstrate that the proposed metasurface can reduce the RCS significantly in an ultra-wide frequency band for both normal and oblique incidences, which makes it promising in the applications such as electromagnetic cloaking.

Optimization of cross-polarization at low radiofrequency fields for sensitivity enhancement in solid-state NMR of membrane proteins reconstituted in magnetically aligned bicelles

Solid-state NMR (ssNMR) of oriented membrane proteins (MPs) is capable of providing structural and dynamic information at nearly physiological conditions. However, NMR experiments performed on oriented membrane proteins generally suffer from low sensitivity. Moreover, utilization of high-power radiofrequency (RF) irradiations for magnetization transfer may give rise to sample heating, thereby decreasing the efficiency of conventional cross-polarization schemes.Here we have optimized the recently developed repetitive cross-polarization (REP-CP) sequence (Tang et al., 2011) to further increase the magnetization transfer efficiency for membrane proteins reconstituted in magnetically aligned bicelles and compared its performance to single-contact Hartmann–Hahn cross-polarization (CP), CP-MOIST and the adiabatic transfer. It has been found that employing the REP-CP sequence at RF amplitudes of 19kHz instead of the commonly used higher RF fields (>45kHz) enhances the efficiency of REP-CP. An additional 30% signal can be obtained as compared to the previously published REP-CP, and 20% when compared to the re-optimized REP-CP at 50kHz RF fields. Moreover, the 15N signal gain of low-power REP-CP was found to be 40% over the adiabatic CP and up to 80% over CP-MOIST. Thus, the low-power REP-CP sequence surpasses all of the previous CP schemes in addition of having the tremendous advantage of reducing the RF powers by a factor of seven, thereby preserving the liquid-like bicelle sample. By contrast, in purely static (NAL crystal) and semi-rigid systems (Pf1 phage), the adiabatic CP was found to be more effective. Periodic oscillations of the intensity profile (distinct from the transient oscillations) as a function of the CP contact time and B1 RF field strengths were observed during the REP-CP optimization with the oscillations becoming more pronounced with lower RF fields. Many-spin simulations were performed to explain the oscillations and their periodicity.

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.

Asymmetric simultaneous phase-inversion cross-polarization in solid-state MAS NMR: Relaxing selective polarization transfer condition between two dilute spins

Double cross polarization (DCP) has been widely used for heteronuclear polarization transfer between 13C and 15N in solid-state magic-angle spinning (MAS) NMR. However, DCP is such sensitive to experimental settings that small variations or deviations in RF fields would deteriorate its efficiency. Here, we report on asymmetric simultaneous phase-inversion cross polarization (referred as aSPICP) for selective polarization transfer between low-γ 13C and 15N spins. We have demonstrated through simulations and experiments using biological solids that the asymmetric duration in the simultaneous phase-inversion cross polarization scheme leads to efficient polarization transfer between 13C and 15N even with large chemical shift anisotropies in the presence of B1 field variations or mismatch of the Hartmann–Hahn conditions. This could be very useful in the aspect of long-duration experiments for membrane protein studies at high fields.

Chemical shift powder spectra enhanced by multiple-contact cross-polarization under slow magic-angle spinning

A simple multiple-contact cross-polarization (CP) scheme is applied to a powder sample of ferrocene and β-calcium formate under static and magic-angle spinning (MAS) conditions. The method is described analytically through the density matrix formalism. We show that multiple equilibrations–re-equilibrations with the proton spin bath improves the polarization transfer efficiency at short contact times and provides higher signal enhancements than state-of-the art techniques such as adiabatic passage through the Hartmann–Hahn condition CP (APHH-CP) when MAS is applied. The resulting chemical shift powder spectra then are identical to the ones obtained by using ROtor-Directed Exchange of Orientations CP (APHH-RODEO-CP) with intensity gains of a factor 1.1–1.3.

Light-emitting-diode-induced fluorescence detection of fluorescent dyes for capillary electrophoresis microchip with cross-polarization method

A cross-polarization scheme is presented to filter out the excitation light from the emission spectrum of fluorescent dyes using green light emitting diodes as a light source and a linear charge coupled device as an intensity detector. The excitation light was linearly polarized and was then used to illuminate the fluorescent dyes in the microchannels of a capillary electrophoresis microchip. The detector was shielded by the second polarizer, oriented perpendicular to the excitation light. The fluorescent signals from Rhodamine B dyes were measured in a dilution series with resulting emission signals and four different concentrations of fluorescent dyes were detected simultaneously with the same excitation source and detector. A limit-of-detection of 1 μM was demonstrated for Rhodamine B dye under the optimal conditions.