Comparison of Methods for Reducing the Effects of Scattering in Spectrophotometry (original) (raw)
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General Chapters 851 SPECTROPHOTOMETRY AND LIGHT SCATTERING
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We investigate potential improvements of continuous-wave diffuse reflectance spectroscopy within highly scattering media by employing polarization gating. Simulations are used to show the extent at which the effective optical pathlength varies in a typical scattering medium as a function of the optical wavelength, the total level of absorption, and the selected polarization channels, including elliptical and circular polarization channels. Experiments then demonstrate that a wavelength dependent polarization gating scheme may reduce the prior knowledge required to solve the problem of chromophore quantification. This is achieved by finding combinations of polarization channels which have similar effective optical pathlengths through the medium at each wavelength.
Modelling and instrumentation for polarized light imaging and spectroscopy of scattering media
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Electronics
The characteristics of modern portable spectrometers based on photodetector arrays make it possible to create on their basis a new class of devices for operational control of the optical properties of various media. The introduction into the practice of diffuse reflectance spectroscopy with spatial resolution is hampered by the lack of an analysis of the influence of the width of the spectral region used and other sources of measurement uncertainty on the unambiguous determination of the optical properties of finely dispersed scattering materials. This article describes a method of determining the coefficient of local diffuse reflection and calculating the spectral parameters of the reduced scattering and absorption of radiation based on the differences in their shape, which are clearly manifested in a wide range of the spectrum. This allows the reduction in the determination of the desired spectral dependencies to the formation of a residual function that requires varying the value...
Microscope spectrometer for light scattering investigations
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We describe a setup including a microscope to study volumes of a few μm 3 by static and dynamic light scattering (DLS) in a backscattering configuration. Light scattered by individual objects of micrometric size can be analyzed in the 400-800 nm spectral range. This setup can also be employed to study both diluted and concentrated colloidal solutions by DLS measurements. For diluted solutions we found evidence of the fluctuations of the number of particles in a confocal volume. We discuss their contribution to the autocorrelation function of the scattered intensity measured as a function of time.
Measurement of Scattering Cross Section with a Spectrophotometer with an Integrating Sphere Detector
Journal of Research of the National Institute of Standards and Technology, 2012
A commercial spectrometer with an integrating sphere (IS) detector was used to measure the scattering cross section of microspheres. Analysis of the measurement process showed that two measurements of the absorbance, one with the cuvette placed in the normal spectrometer position, and the second with the cuvette placed inside the IS, provided enough information to separate the contributions from scattering and molecular absorption. Measurements were carried out with microspheres with different diameters. The data was fitted with a model consisting of the difference of two terms. The first term was the Lorenz-Mie (L-M) cross section which modeled the total absorbance due to scattering. The second term was the integral of the L-M differential cross section over the detector acceptance angle. The second term estimated the amount of forward scattered light that entered the detector. A wavelength dependent index of refraction was used in the model. The agreement between the model and the data was good between 300 nm and 800 nm. The fits provided values for the microsphere diameter, the concentration, and the wavelength dependent index of refraction. For wavelengths less than 300 nm, the scattering cross section had significant spectral structure which was inversely related to the molecular absorption. This work addresses the measurement and interpretation of the scattering cross section for wavelengths between 300 nm and 800 nm.
A new approach to the use of spectroscopic absorbance measurements for anisotropic crystals allows results to be extracted using unpolarized light incident on random crystal orientations. The theory of light propagation in anisotropic absorbing crystals is developed from Maxwell's equations to devise an expression for the transmittance of linearly polarized light traveling in an arbitrary direction in weakly absorbing media. This theory predicts the distribution of transmittance and absorbance as a function of direction and polarization angle of incident light. It is shown how a previously deduced empirical expression, commonly used in infrared spectroscopy, is a good approximation to the full theory under a wide range of conditions. The new theory shows that principal polarized absorbances correspond to the eigenvalues of an absorbance ellipsoid. An expression is derived for the unpolarized absorbance as a function of the angles describing incident light direction, A unpol (ϕ,ψ), and the principal polarized absorbances, A a , A b , A c in an anisotropic crystal A unpol (ϕ,ψ) = ½[A a (cos 2 ϕcos 2 ψ + sin 2 ψ) + A b (cos 2 ϕsin 2 ψ + cos 2 ψ) + A c sin 2 ϕ].
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Circular dichroism (CD) is an important spectroscopic technique for monitoring chirality and biological macromolecule conformation. However, during a CD measurement, absorbance, light scattering/turbidity, and fluorescence can also be detected. The simultaneous measurement of these different spectral features for a single sample is the basis of a multi-mode optical spectrometer. This allows time-efficient gathering of complementary information and provides a scheme to ensure that CD measurements are reliable. Aspects of circular polarization differential light scattering, pH, and temperature variation of a protein (antibody) solution are described. A procedure to help ensure that CD measurements are reliable is described.
Polarization-controlled optimal scatter suppression in transient absorption spectroscopy
Scientific Reports, 2017
Ultrafast transient absorption spectroscopy is a powerful technique to study fast photo-induced processes, such as electron, proton and energy transfer, isomerization and molecular dynamics, in a diverse range of samples, including solid state materials and proteins. Many such experiments suffer from signal distortion by scattered excitation light, in particular close to the excitation (pump) frequency. Scattered light can be effectively suppressed by a polarizer oriented perpendicular to the excitation polarization and positioned behind the sample in the optical path of the probe beam. However, this introduces anisotropic polarization contributions into the recorded signal. We present an approach based on setting specific polarizations of the pump and probe pulses, combined with a polarizer behind the sample. Together, this controls the signal-to-scatter ratio (SSR), while maintaining isotropic signal. We present SSR for the full range of polarizations and analytically derive the optimal configuration at angles of 40.5° between probe and pump and of 66.9° between polarizer and pump polarizations. This improves SSR by 3 3 5 2 ≈. (or 3 compared to polarizer parallel to probe). The calculations are validated by transient absorption experiments on the common fluorescent dye Rhodamine B. This approach provides a simple method to considerably improve the SSR in transient absorption spectroscopy.