Rayleigh Scattering Research Articles

Signal Processing in Optical Frequency Domain Reflectometry Systems Based on Self-Sweeping Fiber Laser with Continuous-Wave Intensity Dynamics

We report on the development of an optical frequency domain reflectometry (OFDR) system based on a continuous-wave Er-doped self-sweeping fiber laser. In this work, we investigate the influence of the input data processing procedure in an OFDR system on the resulting reflectograms and noise level. In particular, several types of signal averaging (in time and frequency domain) and Fourier analysis are applied. We demonstrate that the averaging in the frequency domain can be applied to evaluate absolute values of the local scattering amplitudes related to the Rayleigh light scattering (RLS), which is associated with the interference of scattering signals on microscopic inhomogeneities in optical fibers. We found that the RLS signal remains unchanged in the case of signal averaging in time domain, while the noise floor level decreases by 30 dB with an increasing number of points from 1 to ~450. At the same time, it becomes possible to detect the spectral composition of the scattering at each point of the fiber using windowed Fourier transform. As a result, the sensitivity of the developed system allows us to measure the RLS signal at a level of about 20 dB above the noise floor. The described analysis methods can be useful in the development of distributed sensors based on Rayleigh OFDR systems.

Wavelength tunable double-ring cavity erbium-doped random fiber laser based on a Mach–Zehnder interferometer

A wavelength-tunable double-ring cavity erbium-doped random fiber laser (RFL) based on a Mach–Zehnder interferometer (MZI) was proposed in this study. The MZI structure, which was based on two 2 × 2 optical fiber couplers, exhibits a comb-filtering effect. Rayleigh scattering (RS) was generated by a 10-km-long single-mode fiber (SMF) to provide randomly distributed feedback. A double-ring cavity structure based on forward and backward RS was built using two 2 × 2 fiber couplers. The threshold of the laser was 104 mW, and the slope efficiency of the total power output to the pump power was approximately 1.2%. When the pump power was 158 mW, a laser beam with a wavelength of 1555.3 nm was formed. By adjusting the polarization controller (PC), a single-wavelength switched RFL at 1547.9 nm and 1559.6 nm was obtained. The maximum peak power difference was less than 0.67 dB, the signal-to-noise ratio (SNR) was greater than 17.27 dB, and the peak power fluctuation was less than 0.92 dB during a scan time of 30 min at 25 °C. Regarding dual-wavelength switchable laser emission, two groups of different wavelengths with an SNR greater than 16.88 dB, power fluctuation less than 0.81 dB, and wavelength fluctuation less than 0.26 nm could be achieved under the same monitoring conditions. By further adjusting the PC, three- and four-wavelength RFLs were achieved, and the SNRs exceeded 16.31 dB and 14.64 dB, respectively. The proposed RFL has promising application prospects for distributed sensing systems and speckle imaging.

Nonlinear Optical Materials: Predicting the First-Order Molecular Hyperpolarizability of Organic Molecular Structures

Experimental nonlinear optics (NLO) is usually expensive due to the high-end photonics and electronic devices needed to perform experiments such as incoherent second harmonic generation in liquid phase, multi-photon absorption, and excitation. Nevertheless, exploring NLO responses of organic and inorganic compounds has already opened a world of new possibilities. For example, NLO switches, NLO frequency converters, and a new way to obtain biological images through the incoherent second harmonic generation (SHG) originate from first-order molecular hyperpolarizability (β). The microscopic effect of the coherent or incoherent SHG is, in fact, the β. Therefore, estimating β without using expensive photonic facilities will optimize time- and cost-efficiency to predict if a specific molecular structure can generate light with double its incident frequency. In this work, we have simulated the β values of 27 organic compounds applying density functional theory (PBE0, TPSSh, wB97XD, B3LYP, CAM-B3LYP, and M06-2X) and Hartree–Fock methods using the Gaussian software package. The predicted β was compared with the experimental analogs obtained by the well-known Hyper–Rayleigh Scattering (HRS) technique. The most reliable functionals were CAM-B3LYP and M06-2X, with an unsigned average error of around 25%. Moreover, we have developed post-processing software—Hyper-QCC, providing an effortless, fast, and reliable way to analyze the Gaussian output files.

Facile electrostatic incorporation of the food dye, Dianthine B, for an ultrasensitive tracking and quantification of fexofenadine drug employing an ''on-on++ fluorescence'' strategy

This research presents a facile spectrofluorimetric technique for detecting fexofenadine (FXD) drug based on the idea of Dianthine B's Resonance Rayleigh Scattering response augmentation. A food dye, Dianthine B (DB), is a fluoresine base-structured pigment used as a food additive, flavor, bio-stainer, and for a medicational prognosis purpose. Interest in employing food additives for tagging and tracking has increased because of their safety, elevated quantum outputs, and propensity to biomaterials labeling. The planned method validated and evaluated the target drug quickly and efficiently using a direct, facile, simple, and single-pot-single-step spectrofluorimetric approach, making it more favorable to the environment. Facile coupling between FXD and Dianthine B occurred rapidly in an acidic environment. A correlation was established between the increased responsiveness of the food dye to Resonance Rayleigh Scattering signals as a result of FXD and the molecular size of the coupled product formed at 361 nm. The linear range was determined to be from 50 to 1500 ng mL−1, while the sensitivity limits were 15 ng mL−1 for LOD and 46 ng mL−1 for LOQ. Analyte-dye coupled products were studied to establish the optimal settings for the system's tunable variables. Additionally, there were no problems with the system meeting the International Council for harmonisation ICH requirements. Plus, enough of the material was retrieved for medicinal applications. The coupled product's quantum yield was evaluated. Additionally, the environmental evaluation of the proposed system was done using ecological metrics, including an eco-scale (95/100), the National Environmental Methods Index, NEMI (full green pictograms), the Green Analytical Procedure Index (GAPI), and the Analytical GREEnness Measure, AGREE (0.8/1). These results indicated that the planned method's significant level of greenness was achieved.

A novel second-order calibration algorithm for processing fluorescence data with scattering: Three-direction resection ATLD

The light scattering is common during fluorescence measurements, such as first- and second-order Rayleigh and Raman scatterings, which are not conducive to the decomposition of the multi-way data because it destroys the multi-linear structure. This paper proposed two second-order calibration algorithms, including three-direction resection alternating trilinear decomposition (TDR-ATLD) and three-direction resection self-weight alternating trilinear decomposition (TDR-SWATLD), which are based on an improved trilinear decomposition model for directly decomposition of the three-way fluorescence data with scattering regions removed. Through the simulation data, the proposed algorithms were compared with other three algorithms (ATLD, Interpolation-ATLD, TDR-PARAFAC). The calculation results of three algorithms (TDR-PARAFAC, TDR-ATLD and TDR-SWATLD) are better at small or large scattering widths. Both TDR-ATLD and TDR-SWATLD are insensitive to the number of components and the performance of TDR-ATLD had little fluctuation with different noise levels among the five algorithms. Additionally, TDR-ATLD and TDR-SWATLD are better than other algorithms when dealing with collinear data. The proposed algorithms were also used in two sets of real data, compared with other two algorithms (Interpolation-ATLD and TDR-PARAFAC) for dealing with scattering, the proposed algorithms could deal with fluorescence data with scattering and provided satisfactory results and TDR-ATLD has faster convergence. In general, TDR-ATLD has greater potential and performs better in processing fluorescence data with scattering.

The effect of novel antihypertensive drug valsartan on lysozyme aggregation: A combined in situ and in silico study

Protein misfolding can result in amyloid fiber aggregation, which is associated with various types of diseases. Therefore, preventing or treating abnormally folded proteins may provide therapeutic intervention for these diseases. Valsartan (VAL) is an angiotensin II receptor blocker (ARB) that is used to treat hypertension. In this study, we examine the anti-aggregating effect of VAL against hen egg-white lysozyme (HEWL) amyloid fibrils through spectroscopy, docking, and microscopic analysis. In vitro formation of HEWL amyloid fibrils was indicated by increased turbidity, RLS (Rayleigh light scattering), and ThT fluorescence intensity. 10 μM VAL, amyloid/aggregation was inhibited up to 83% and 72% as measured by ThT and RLS respectively. In contrast, 100 μM VAL significantly increases the fibril aggregation of HEWL. CD spectroscopy results show a stabilization of HEWL α-helical structures in the presence of 10 μM VAL while the increase in β-sheet was detected at 100 μM concentration of VAL. The hydrophobicity of HEWL was increased at 100 μM VAL, suggesting the promotion of aggregation via its self-association. Steady-state quenching revealed that VAL and HEWL interact spontaneously via hydrogen bonds and van der Waals forces. Transmission electron microscopy (TEM) images illustrate that the needle-like fibers of HEWL amyloid were reduced at 10 μM VAL, while at 100 μM the fibrils of amyloid were increased. Additionally, our computational studies showed that VAL could bind to two binding sites within HEWL. In the BS-1 domain of HEWL, VAL binds to ASN59, ILE98, ILE58, TRP108, VAL109, SER50, ASP52, ASN59, ALA107, and TRP108 residues with a binding energy of −9.72 kcal mol−1. Also, it binds to GLU7, ALA10, ALA11, CYS6, ARG128, and ARG14 in the BS-2 domain with a binding energy of −5.89 kcal mol−1. VAL, therefore, appears to have dual effect against HEWL aggregation. We suggest that VAL stabilizes HEWL's aggregation-prone region (APR) at 10 μM, preventing aggregation. Also, we assume that at 100 μM, VAL occupies BS-2 beside BS-1 and destabilizes the folding structure of HEWL, resulting in aggregation. Further studies are needed to investigate the mechanism of action and determine its potential side effects.

Designing a unique molecular size-dependent approach for determining levamisole via synergizing Rayleigh Scattering response of Cilefa Pink B dye; application to bulk, dosage forms, and biofluids; method greenness evaluation

Levamisole is an anti-helminthic agent employed medically to treat worm-causative disease, also used as an immune system modifying agent in rheumatic arthritis and as assistant therapy for treating the cancers of the head, colorectal, and neck regions. The first innovative size-dependent approach for levamisole drug analysis is described in this study. It is both efficient and environmentally benign. The drug was evaluated and validated using a green, one-pot, and direct size-dependent technique in this approach. An instant complex was created by combining levamisole and Cilefa Pink B in a mildly acidic medium. The fluorimetric investigation was based on the augmentation effect of levamisole on the Rayleigh scattering response amplitude of a biological dye (Cilefa Pink B) at 424 nm. which was related to produced complex's molecular size. Linearity ranged from 0.08 to 2.0 µg mL−1, with sensitivity limits of 0.023 and 0.069 µg mL−1, respectively. Analytical modulation of Levamisole-Cilefa Pink B complexes was done for all system parameters. In addition, the system was found to meet ICH criteria. Moreover, this technique successfully recovered the substance in the prescribed medicinal dosage forms. The created fluorimetric technique was also successfully employed to track the drug of interest in human biofluids, which was a significant accomplishment. Finally, an eco-scale was applied to recognize the designed method's environmental friendliness.

High-Spatial-Resolution Strain Sensor Based on Rayleigh-Scattering-Enhanced SMF Using Direct UV Exposure

A high-spatial-resolution, i.e., 2.0 mm, strain sensor based on a Rayleigh-scattering-enhanced SMF, i.e., exposed SMF (E-SMF) with direct UV laser, was experimentally demonstrated. The enhancement of Rayleigh-scattering (RS) intensity could be tuned by adjusting the exposure parameters, i.e., distance between SMF and Phase mask, energy of UV laser and velocity of the SMF. A 1.0 m long E-SMF with a RS enhancement of 37.3 dB was obtained, where the exposure time was only 100 s. Compared with the un-exposed SMF (UE-SMF), the strain profiles of the E-SMF could be clearly demodulated without fluctuation at a spatial resolution of 2.0 mm using traditional cross-correlation algorithm when the applied strain was from 200 to 2600 μϵ.

Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study.

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 °C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in α-helix content and β-sheet content between at 10 µM CTZ and 100 µM. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN57, ASP52, TRP108, TRP63, TRP63, ILE58, and ALA107 residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol-1. We suggest that at 10 µM and 100 μM, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Optical Sensing of Toxic Cyanide Anions Using Noble Metal Nanomaterials.

Water toxicity, one of the major concerns for ecosystems and the health of humanity, is usually attributed to inorganic anions-induced contamination. Particularly, cyanide ions are considered one of the most harmful elements required to be monitored in water. The need for cyanide sensing and monitoring has tempted the development of sensing technologies without highly sophisticated instruments or highly skilled operations for the objective of in-situ monitoring. Recent decades have witnessed the growth of noble metal nanomaterials-based sensors for detecting cyanide ions quantitatively as nanoscience and nanotechnologies advance to allow nanoscale-inherent physicochemical properties to be exploited for sensing performance. Particularly, noble metal nanostructure e-based optical sensors have permitted cyanide ions of nanomolar levels, or even lower, to be detectable. This capability lends itself to analytical application in the quantitative detection of harmful elements in environmental water samples. This review covers the noble metal nanomaterials-based sensors for cyanide ions detection developed in a variety of approaches, such as those based on colorimetry, fluorescence, Rayleigh scattering (RS), and surface-enhanced Raman scattering (SERS). Additionally, major challenges associated with these nano-platforms are also addressed, while future perspectives are given with directions towards resolving these issues.

Utilizing Rayleigh light scattering of anthracene nanoparticles for determination of p-cresol in exhaled breath condensate

A simple and sensitive Rayleigh light scattering (RLS) method is developed for the quantification of p-cresol in exhaled breath condensate (EBC). The method is based on an aggregation of anthracene nanoparticles in acidic media after adding the p-cresol which results in a marked enhancement in RLS intensity at 410 nm. The increase of RLS intensity is directly proportional to the p-cresol concentration, and the linearity is obtained in the range of 0.007–0.5 μg mL−1, with the LOD of 0.004 ng mL−1. The relative standard deviations (RSDs, n = 5) for inter-day and intra-day analysis were <6.5 % for investigated concentrations. Finally, the validated method is applied for the analysis of p-cresol in EBC samples taken from patients suffering chronic kidney disease.

Multi-Parameter Measurement Method of Single Mode Fiber Based on Bidirectional Rayleigh Scattering

Multi-Parameter Measurement Method of Single Mode Fiber Based on Bidirectional Rayleigh Scattering

Investigation of 10 kHz Filtered Rayleigh Scattering and CH2O Planar Laser-Induced Fluorescence Measurement in Two-Stage Swirl Combustor

Investigation of 10 kHz Filtered Rayleigh Scattering and CH₂O Planar Laser-Induced Fluorescence Measurement in Two-Stage Swirl Combustor

Investigation of first hyper-polarisability molecular switches between enol–keto equilibrium of phenyl benzodifurantrione: a DFT-based computational study

The contrast of the second-order nonlinear optical response between the enol (anti and syn) and keto tautomeric equilibrium of 7-Phenyl-7H-benzo[1,2-b;4,5-b’]difuran-2,3,6-trione (PBDT) with donor/acceptor substitution at the para position of phenyl group have been computed involving electron correlation, frequency dispersion, and solvent polarity effects. The Hyper Rayleigh Scattering (HRS) first-hyperpolarisability contrast between the enol and keto forms for PBDT and Nitro-substituted PBDT is 3.5 and 2.8, respectively. However, in NMe2-substituted PBDT, a large contrast (12 times) of HRS first hyper-polarisability between enol (anti and syn) and keto form is observed. Present study reveals that electron donor group (–NMe2) in PBDT plays a pivotal role for obtaining very high contrast in the first hyper-polarisability (β HRS and β vec) values between the enol and keto forms compare to the electron acceptor (–NO2) group substitution at the same position. The solvent effect also plays a significant role in further improving the first hyper-polarisability value and its switching behaviour between the enol and keto forms of NMe2-PBDT. NLO switching contrast between the enol and keto forms of donor/acceptor substituted PBDT is also explained using the electronic features including UV-Vis absorption spectra, HOMO–LUMO electron densities, optical energy gaps, charge transfer contribution etc.

Circular Polarimetry of Hard X-rays with Rayleigh Scattering

We present a theoretical investigation of the elastic Rayleigh scattering of X-rays by atomic targets. Special attention is paid to the question of how the polarization of the scattered photons is affected if the incident light is itself polarized. In particular, we found that the circular polarization of the incoming X-rays may lead to a remarkable modification of the linear polarization of the scattered photons. Based on this ’circular-to-linear-polarization-transfer’ and on the fact that the linear polarization of X-rays can be conveniently observed by solid-state Compton detectors, we argue that Rayleigh scattering may be used as a tool for circular polarimetry of hard X-rays. To illustrate our proposal, we performed detailed calculations of 145 and 500 keV circularly polarized photons scattered by lead atoms. Based on these calculations, we found that the photon scattering under large angles with respect to the incident beam direction is most favorable for the circular polarimetry of hard X-rays. In particular, for 500 keV photon energy and scattering angles around 70 deg we found a remarkable modification of the linear polarization of scattered light for the case when the incident radiation is circularly polarized.

Stimulated Rayleigh Scattering Enhanced by a Longitudinal Plasma Mode in a Periodically Driven Dirac Semimetal Cd_{3}As_{2}.

Using broadband (12-45THz) multi-terahertz spectroscopy, we show that stimulated Rayleigh scattering dominates the transient optical conductivity of cadmium arsenide, a Dirac semimetal, under an optical driving field at 30THz. The characteristic dispersive line shape with net optical gain is accounted for by optical transitions between light-induced Floquet subbands, strikingly enhanced by the longitudinal plasma mode. Stimulated Rayleigh scattering with an unprecedentedly large refractive index change may pave the way for slow light generation in conductive solids at room temperature.

Using Global Existing Fiber Networks for Environmental Sensing

We review recent advances in distributed fiber optic sensing (DFOS) and their applications. The scattering mechanisms in glass, which are exploited for reflectometry-based DFOS, are Rayleigh, Brillouin, and Raman scatterings. These are sensitive to either strain and/or temperature, allowing optical fiber cables to monitor their ambient environment in addition to their conventional role as a medium for telecommunications. Recently, DFOS leveraged technologies developed for telecommunications, such as coherent detection, digital signal processing, coding, and spatial/frequency diversity, to achieve improved performance in terms of measurand resolution, reach, spatial resolution, and bandwidth. We review the theory and architecture of commonly used DFOS methods. We provide recent experimental and field trial results where DFOS was used in wide-ranging applications, such as geohazard monitoring, seismic monitoring, traffic monitoring, and infrastructure health monitoring. Events of interest often have unique signatures either in the spatial, temporal, frequency, or wavenumber domains. Based on the temperature and strain raw data obtained from DFOS, downstream postprocessing allows the detection, classification, and localization of events. Combining DFOS with machine learning methods, it is possible to realize complete sensor systems that are compact, low cost, and can operate in harsh environments and difficult-to-access locations, facilitating increased public safety and smarter cities.

Measurements of Rayleigh–Brillouin scattering spectra of Ar, N[formula omitted], and CH[formula omitted] at pressures up to 20 atm

This paper describes the spectral measurement of Rayleigh–Brillouin scattering (RBS) in Ar, N2, and CH4 at 300 K and pressures up to 20 atm, which is relevant for the application of filtered Rayleigh scattering (FRS) diagnostics at elevated pressure. The measurement scheme for RBS spectra was relatively novel, based on an air-spaced virtually imaged phased array (VIPA), capable of rapid acquisition at high resolution. However, the VIPA’s nonuniform dispersion, irregular instrument response function, and sensitivity to local ambient conditions challenged the reconstruction of high-fidelity lineshapes in frequency space. Nonuniform dispersion was addressed using the VIPA dispersion function, and sensitivity of the air-spaced optic to the refractive index of air was minimized by tracking local temperature and pressure during measurements. Irregular instrument response was remedied with a computational model of the VIPA’s optical field, and by deconvolution. A comparison of experimental lineshapes with model predictions by Tenti S6 yielded good agreement. In the kinetic and nominal transition regimes, local residuals were within 3% for Ar and N2, and 5–10% for CH4. Beyond the transition regime, experimental lineshapes continued to display good qualitative agreement, with frequency shifts of the Brillouin peaks representing the main deviation from model prediction. Artifacts in the experimental data from uncertainty and the deconvolution algorithm were identified and discussed. This work establishes an experimental foundation for future measurements of RBS spectra, which will assist in the development of FRS diagnostics of reacting flows at high pressure.

Research on the Measurement Accuracy of Shipborne Rayleigh Scattering Lidar

This paper aims to study the measurement accuracy of Rayleigh scattering lidar (light detection and ranging) based on a ship platform and analyze the influence of the laser beam uncertainty on the temperature inversion results. Taking the ship platform roll data as a reference, the Rayleigh scattering lidar oscillating model is simplified to a sine function, and the inversion accuracy of atmospheric temperature is analyzed under different settled observation angles and different roll angles. When the settled observation angle is 0° and the roll angle amplitudes are 10°, 20°, and 30°, the maximum deviations of the temperature within the height range of 30–80 km are 3.47 K, 13.73 K, and 22.78 K, respectively, and the average deviations are 2.35 K, 9.09 K, and 12.95 K, respectively. When the observation angle is set to 30° and the roll angle amplitudes are 10°, 20°, and 30°, the maximum deviations of the temperature within the height range of 30–80 km are 11.75 K, 27.49 K, and 53.50 K, respectively, and the average deviations are 11.05 K, 13.88 K, and 16.12 K, respectively. The results of this paper show that ship platform rolling greatly influences the measurement of atmospheric temperature, which provides a certain data reference for the construction and use of Rayleigh scattering lidar in the ship platform.

Analyzing protein conjugation reactions for antibody-drug conjugate synthesis using polarized excitation emission matrix spectroscopy.

Antibody-drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug-to-antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in-process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing.Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, Rh ) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with Rh (R2 = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in-process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi-attribute test method for ADC manufacturing.