Exploring the hidden depth by confocal Raman experiments with variable objective aperture and magnification.

The article analyzes experimentally and theoretically the influence of microscope parameters on the pinhole-assisted Raman depth profiles in uniform and composite refractive media. The main objective is the reliable mapping of deep sample regions. The easiest to interpret results are found with low magnification, low aperture, and small pinholes. Here, the intensities and shapes of the Raman signals are independent of the location of the emitter relative to the sample surface. Theoretically, the results can be well described with a simple analytical equation containing the axial depth resolution of the microscope and the position of the emitter. The lower determinable object size is limited to 2-4 µm. If sub-micrometer resolution is desired, high magnification, mostly combined with high aperture, becomes necessary. The signal intensities and shapes depend now in refractive media on the position relative to the sample surface. This aspect is investigated on a number of uniform and stacked polymer layers, 2-160 µm thick, with the best available transparency. The experimental depth profiles are numerically fitted with excellent accuracy by inserting a Gaussian excitation beam of variable waist and fill fraction through the focusing lens area, and by treating the Raman emission with geometric optics as spontaneous isotropic process through the lens and the variable pinhole, respectively. The intersectional area of these two solid angles yields the leading factor in understanding confocal (pinhole-assisted) Raman depth profiles. Spearfishing is a well-known example of the effects of refraction at the boundary between two index-mismatched media. The object Greal is seen, due to refraction, as Gvir from the angle ß (without knowing the depth position). The real position is obtained under the angle α. In a microscope (see inset), index mismatch deforms the image point of Greal into an image line. The pinhole substantially reduces deformations and allows the determination of the position of the point emitter G. (Cartoon designed by Sofia Anker).

Pigmentation of White, Brown, and Green Chicken Eggshells Analyzed by Reflectance, Transmittance, and Fluorescence Spectroscopy.

We report on the reflectance, transmittance and fluorescence spectra (λ=200-1200 nm) of four types of chicken eggshells (white, brown, light green, dark green) measured in situ without pretreatment and after ablation of 20-100 µm of the outer shell regions. The color pigment protoporphyrin IX (PPIX) is embedded in the protein phase of all four shell types as highly fluorescent monomers, in the white and light green shells additionally as non-fluorescent dimers, and in the brown and dark green shells mainly as non-fluorescent poly-aggregates. The green shell colors are formed from an approximately equimolar mixture of PPIX and biliverdin. The axial distribution of protein and colorpigments were evaluated from the combined reflectances of both the outer and inner shell surfaces, as well as from the transmittances. For the data generation we used the radiative transfer model in the random walk and Kubelka-Munk approaches.

Comparative Raman study of transparent and turbid materials: models and experiments in the remote sensing mode.

The influence of turbidity on the Raman signal strengths of condensed matter is theoretically analyzed and measured with laboratory - scale equipment for remote sensing. The results show the quantitative dependence of back- and forward-scattered signals on the thickness and elastic-scattering properties of matter. In the extreme situation of thin, highly turbid layers, the measured Raman signal strengths exceed their transparent analogs by more than a factor of ten. The opposite behavior is found for thick layers of low turbidity, where the presence of a small amount of scatterers leads to a decrease of the measured signal. The wide range of turbidities appearing in nature is experimentally realized with stacked polymer layers and solid/liquid dispersions, and theoretically modeled by the equation of radiative transfer using the analytical diffusion approximation or random walk simulations. Graphical abstract Spatial Raman emission profiles in transparent and turbid materials.

Penetration of light into multiple scattering media: model calculations and reflectance experiments. Part II: The radial transfer.

In continuation of our contribution to "The Axial Transfer" (Appl. Spectr. 2012. 66(8): 934-943), this paper describes the distribution of localized incident radiation in multiple scattering layers of arbitrary thickness and analyzes the lateral intensity profiles of radiation leaving the sample from its illuminated and non-illuminated surfaces. The theoretical profiles are calculated with different approximations of the equation of transfer. We derive for both non-absorbing and absorbing layers simple analytical expressions and verify their accuracy and range of applicability by comparison with Monte Carlo simulations. Particular emphasis is given to the analysis of the radial absorption, an under-theorized and under-investigated feature that can help to identify weak or hidden absorbers. In addition, we contribute to the description of how the radial reflectance is affected by anisotropy or by error sources like multiple surface reflection for samples in glass cells or deflectance (sideway loss) of radiation in small samples. Finally, the theoretical results are compared with experimental data of radial reflectance for quasi non-absorbing and absorbing powder layers.

Quantitative Raman spectroscopy in turbid matter: reflection or transmission mode?

Raman intensities from reflection (X(R)) and transmission (X(T)) setups are compared by calculations based on random walk and analytical approaches with respect to sample thickness, absorption, and scattering. Experiments incorporating strongly scattering organic polymer layers and powder tablets of pharmaceutical ingredients validate the theoretical findings. For nonabsorbing layers, the Raman reflection and transmission intensities rise steadily with the layer thickness, starting for very thin layers with the ratio X(T)/X(R) = 1 and approaching for thick layers, a lower limit of X(T)/X(R) = 0.5. This result is completely different from the primary irradiation where the ratio of transmittance/reflectance decays hyperbolically with the layer thickness to zero. In absorbing materials, X R saturates at levels that depend strongly on the absorption and scattering coefficients. X T passes through a maximum and decreases then exponentially with increasing layer thickness to zero. From the calculated radial intensity spreads, it follows that quantitative transmission Raman spectroscopy requires diameters of the detected sample areas be about six times larger than the sample thickness. In stratified systems, Raman transmission allows deep probing even of small quantities in buried layers. In double layers, the information is independent from the side of the measurements. In triple layers simulating coated tablets, the information of X T originates mainly from the center of the bulk material whereas X R highlights the irradiated boundary region. However, if the stratified sample is measured in a Raman reflection setup in front of a white diffusely reflecting surface, it is possible to monitor the whole depth of a multiple scattering sample with equal statistical weight. This may be a favorable approach for inline Raman spectroscopy in process analytical technology.

Penetration of light into multiple scattering media: model calculations and reflectance experiments. Part I: the axial transfer.

The article presents two general equations of radiation penetration into layers of diffuse reflectors. One of the equations describes the depth origins of reflection, the other the depth profiles of absorption. The equations are evaluated within the theory of radiative transfer applying various degrees of analytical approximations and Monte Carlo simulations. The data are presented for different scattering and absorption coefficients, arbitrary layer thicknesses, collimated and diffused irradiation, and anisotropic forward scattering. The calculated mean depths of reflection are always lower than the mean depths of absorption. For nearly non-absorbing layers, the mean depths of absorption are about one third of the physical layer thickness. In contrast, penetration saturates for strong absorbers at very low depth levels. From the simulated data, methods are derived for the determination of the penetration depth from reflectance and transmittance data of thin layers or from radially diffused reflectance profiles upon spot irradiation. The methods are experimentally verified for a series of metal oxide powders with particle sizes ranging from much smaller to much larger than the wavelength of irradiation and for microcrystalline cellulose stained with different concentrations of an organic dye.

Dark-field scattering microscopy for spectral characterization of polystyrene aggregates.

Light scattering measurements of particle aggregates contain complex information which is difficult to decrypt. Dark-field scattering microscopy in the visible range is used to characterize multi-arranged polystyrene beads. First, measured light scattering spectra of single spheres are compared with the Mie theory. Then, additional spectral measurements of three different sample sets of sphere aggregates are carried out. The aggregates consist of homogeneous spheres and differ in number of spheres, arrangement and contact area. Principal component analysis is used to reduce the number of variables and achieve an accurate classification regarding the aggregate characteristics.

Using scattering and absorption spectra as MCR-hard model constraints for diffuse reflectance measurements of tablets.

One of the most often used tools in process analytical technology (PAT) is NIR spectroscopy as a non-destructive fast and reliable method to identify and quantify active pharmaceutical ingredients (API) in tablets. Very little work has been devoted to analyse the effects of scatter on quantitative analysis of the chemical composition. A novel approach to compensate scatter in reflectance spectroscopy which is more science based will be presented here. The basic assumption is to determine in step 1 a separate scattering spectral fingerprint, denoted as S spectra, and an absorption spectral fingerprint, denoted as K spectra. In the second step, the two spectra may then be used as input to the alternating least square (ALS) algorithm in multivariate curve resolution (MCR) in order to account for the spectral distortions due to the interaction of scatter and absorption. Standard tablets with a mass of 1.5 g and a diameter of 20 mm (thickness approx. 3.4 mm, optically infinite) were prepared according to a central composite design by mixing theophylline, magnesium stearate and cellactose at three different compactions of 31, 156 and 281 MPa. The samples are measured by an UV/Vis/NIR spectrometer attached with an integrating sphere in the wavelength range from 500 up to 2100 nm. The diffuse reflectance spectra of the center point sample with an optically infinite thickness R(infinity) as well as a sample of finite thickness R(0) ("optically thin") is measured as reference for the S and K spectra which are then calculated with the exponential solution of the Kubelka-Munk equation. After normalization, the S spectrum and the K spectrum of a single tablet are integrated as hard model constraints into the MCR-ALS procedure. In comparison to PLS modeling with EMSC pretreatment of the spectra, the hard model constrained MCR-ALS algorithm results in an improved prediction of the concentration of the API together with a higher robustness of the calibration models.

Design of pi-conjugated organic materials for one-dimensional energy transport in nanochannels.

Various end-substituted distyrylbenzenes have been synthesized to serve as guest molecules in inclusion compounds to promote efficient energy transport along one-dimensional channels. Their optical and photophysical properties have been characterized at both experimental and theoretical levels. All molecules display a large transition dipole moment between the ground state and lowest excited state and hence a short radiative lifetime (on the order of 1-2 ns). They also exhibit a large spectral overlap between the emission and absorption spectra, which enables efficient energy transport between molecules arranged in a head-to-tail configuration in nanochannels. Hopping rates on the order of 10(12) s(-1) are calculated at a full quantum-chemical level; this is much larger than the radiative lifetimes and opens the way for energy migration over large distances. Changes in the nature of the terminal substituents are found to modulate the optical properties weakly but to impact significantly the energy transfer rates.

Spatially Resolved Immobilization of Peptides by Electrochemical Polymerization after Photolytic Cleavage of a Protecting Group.

Microstructuring of surfaces: Electrochemical polymerization after removal of a photolabile protecting group (nitrobenzyl group) represents a new method for spatially resolved immobilization of ligands or receptors. Thus, the electropolymerization of 3-hydroxyphenylacetyl peptides such as 1 on electrodes can be controlled by light.

Investigating the Selectivity of Triacontyl Interphases.

What causes the shape selectivity of C30 phases? This question can be answered by combining NMR and fluorescence spectroscopies with HPLC separations at various temperatures. The selectivities depend on the ratio of trans to gauche conformations of the alkyl chains, whose dynamic behavior was characterized with a two-dimensional solid-state NMR spectrum (shown on the right).