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.

AbstractWe 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.

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.

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.

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.

The present Perspective critically re-examines the photophysics of para-distyrylbenzene (DSB) as a prototype of herringbone-arranged H-aggregates to resolve the apparent contradiction of the frequently reported "aggregation-induced emission quenching" in H-aggregates on one side and highly emissive DSB crystals on the other and discusses the signatures and fate of excitons in single- and polycrystalline samples, including size and polarization effects.

We describe the design and performance of a Hyperspectral Imaging System (HSI) for label-free characterization of human metaphase chromosomes. Chromosomes consist of a DNA-protein complex that is organized in sub-structures and can be described by an array of "particles" with different size and refractive indices. This locally resolved stray light pattern can be used to visualize and characterize unstained chromosomes. The paper describes an imaging system where stray light spectra of chromosomes are detected using a Pushbroom Imaging device attached to a standard microscope in combination with multivariate data analysis. To proof the concept, single particle spectra as well as particle array spectra are analyzed and explained by means of Mie scattering theory and the results are confirmed with FDTD (Finite Difference Time Domain) simulations. This label free signature is due to the superposition of the interference pattern of the different layer thicknesses, the spectral interference of the band pattern, changes in refractive indices along the chromosome axis as well as the absorption of chromophores in different spectral regions of the chromatin condensation. This complex spectral signature can be analyzed by means of a principal component analysis (PCA) and classified in a multidimensional PCA space.

The adsorption equilibria of proton accepting and donating analytes on porous silica beads applicable as stationary chromatographic phases were investigated by UV/vis absorption spectroscopy. Fluorescence spectroscopy was utilized to characterize the nature of the species formed at the silica surfaces after adsorption. In order to control the equilibria and states of adsorption the active silanol surface centers were partly shielded by adsorbed water or by two types of polymeric coatings, (i) polymerized 1,4-divinylbenzene (DVB) with loadings of 200 mg and 500 mg DVB/g silica, respectively, (ii) polymerized N,N'-diallyl-L-tartardiamide bis-(4-tertbutylbenzoate) (TBB) with a loading of 135 mg TBB/g silica. Acridine orange, 1,2,7,8-dibenzacridine, 3,4,5,6,-dibenzacridine, and lumichrome were used as fluorescent analytes with proton accepting or donating nitrogen centers. The fluorescence anisotropies show that the adsorbed species at the uncoated silica surface are highly immobilized. Coating considerably reduces the equilibrium constants of adsorption. polymerized N,N'-diallyl-L-tartardiamide bis-(4-tertbutylbenzoate) works much better than polymerized 1,4-divinylbenzene. In the latter case a large amount of polymer is necessary in order to produce a significant effect. (C) 2011 Elsevier B.V. All rights reserved.

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.

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 1.5g and a diameter of 20mm (thickness approx. 3.4mm, optically infinite) were prepared according to a central composite design by mixing theophyllin, magnesiumstearate and cellactose at three different compactions of 31, 156 and 281MPa. The samples are measured by an UV/Vis/NIR spectrometer attached with an integrating sphere in the wavelength range from 500 up to 2100nm. The diffuse reflectance spectra of the center point sample with an optically infinite thickness R∞ as well as a sample of finite thickness R0 (“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.

Partially deuterated 1,4-distyrylbenzene () is included into the pseudohexagonal nanochannels of perhydrotriphenylene (PHTP). The overall and intramolecular mobility of is investigated over a wide temperature range by (13)C, (2)H NMR as well as fluorescence spectroscopy. Simulations of the (2)H NMR spectral shapes reveal an overall wobble motion of in the channels with an amplitude of about 4 degrees at T = 220 K and 10 degrees at T = 410 K. Above T = 320 K the wobble motion is superimposed by localized 180 degrees flips of the terminal phenyl rings with a frequency of 10(6) Hz at T = 340 K. The activation energies of both types of motions are around 40 kJ mol(-1) which imply a strong sterical hindrance by the surrounding PHTP channels. The experimental vibrational structure of the fluorescence excitation spectra of is analyzed in terms of small amplitude ring torsional motions, which provide information about the spatial constraints on by the surrounding PHTP host matrix. Combining the results from NMR and fluorescence spectroscopy as well as of time-dependent density functional calculations yields the complete potential surfaces of the phenyl ring torsions. These results, which suggest that intramolecular mobility of is only reduced but not completely suppressed by the matrix, are corroborated by MD simulations. Unrealistically high potential barriers for phenyl ring flips are obtained from MD simulations using rigid PHTP matrices which demonstrate the importance of large amplitude motions of the PHTP host lattice for the mobility of the guest molecules.

Elementary photoexcitations in polycrystalline films of oligophenylenevinylenes (OPV) are studied by pump-probe spectroscopy with sub-30 fs time resolution. When pumping 0.9 eV above the absorption edge, we find that predominantly charge carriers are formed as primary photoexcitations, whereas the majority of the neutral singlet excited states are formed in a secondary process upon geminate charge recombination. The sublinear pump intensity dependence of singlet states concentration is explained by ultrafast annihilation involving hot singlet states. Upon pumping at the absorption edge, instantaneous stimulated emission is observed instead of charged states absorption, demonstrating that singlet states are the predominant primary photoexcitations at low excess energies.

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.

Using electronic absorption and fluorescence spectroscopic techniques, as well as quantum chemical calculations, we have studied the electronic spectra of thia-bridged stilbenophane (TSP) with close cofacial contact of two trans-stilbene (t-SB) units. Compared to the t-SB monomer, the experimental consequences of the cofacial arrangement are (i) a splitting of the main absorption band with a weakly allowed emitting state, and (ii) a strongly red-shifted, unstructured emission spectrum with long fluorescence decay times. According to the theoretical investigations, the two t-SB units are strongly bent in the electronic ground state (SO), because of repulsive pi-pi overlap. In the first excited state (S I), the t-SB units become almost planar, because of attractive pi*-pi* overlap. As a consequence, the symmetry-forbidden S-0 <----> S-1 transition couples strongly to interchromophore breathing modes of low frequency (v(1) = 67 cm(-1), V-2 = 117 cm(-1)), yielding structureless spectra with large Stokes shifts. The features of the calculated spectra are in good agreement with the experimental data. The results indicate that strong intermolecular vibronic coupling is also responsible for "excimer-like" emission in organic molecular crystals of cofacially arranged molecules. Furthermore, the different geometries in the S-0 and S-1 states of TSP give evidence for the mechanism of [2+2]photodimerization of t-SB in solutions.

Oxygen causes reversible and irreversible detrimental effects to the performance of organic (opto-)electronic devices. In order to get some insight into the mechanisms of these effects, we investigated the kinetics of fluorescence quenching (FQ) in thin films (d≈100 nm–12 μm) of regioregular polyalkylthiophenes upon exposure to oxygen.The kinetics of FQ consists of a fast component, which is fully reversible, as well as a slow component, which is partially irreversible. The fast reversible component leads to a loss of fluorescence intensity of approximately 2% at an oxygen partial pressure of 1 bar within milliseconds. It is independent of the intensity of the exciting light and is ascribed to collisional quenching of excited singlet states after oxygen diffusion into the bulk of the film. The diffusion coefficients of oxygen and singlet excitons were determined as D(O2)=1.5(3)×10−7 cm2s−1 and D(S1)=5×10−4 cm2s−1, respectively. The slow reversible component, whose amplitude depends on light intensity, occurs on a time-scale of minutes. It is assigned to the formation of charge-transfer complexes between excited singlet states of polythiophene and oxygen. Femtosecond pump-probe experiments show that effective quenching of excited singlet states by oxygen takes place on a picosecond time-scale, leading to the enhanced formation of charged states.

Quantum-chemical calculations have been performed to characterize the potentiality of recently synthesized end-substituted oligophenylenevinylenes (OPVs) as excitation shuttles and to design more efficient derivatives. The approach provides quantitative estimates of the parameters controlling the exciton transfer rates and strategies to promote directional energy transfer.

We analyze the generation and recombination of charged states in oligo(phenylenevinylene)s at high photoexcitation densities, using femtosecond pump-probe spectroscopy as a direct probe for the time-dependent density of photogenerated singlet, triplet, and doublet states. Pairs of charged states are formed during the exciting laser pulse, partly by a two-photon process. Geminate recombination of these pairs is found in the first ps after formation, after which a nongeminate dispersive mechanism prevails. The application of high electric field strengths opens up an additional channel for charge carrier formation, involving field-induced electron transfer between two singlet excitons. This process is competing with bimolecular singlet exciton annihilation. Field-induced formation of triplet states is governed by nongeminate recombination of field-induced charged states. We give values for cross sections of the photogenerated species, the efficiency of field-induced charge carrier photogeneration and for the early charge carrier mobility.

The optical properties, molecular orientations and host-guest ratios of a series of p,p'-disubstituted oligophenylenevinylenes (OPVs), incorporated in the nanochannels of perhydrotriphenylene, are investigated by polarized fluorescence and UV absorption spectroscopy. The spectral positions and fine-structures of the fluorescence bands are very similar to those obtained in solution, thus indicating only weak interactions within the channels. In three-dimensional arrangements, the spectra are additionally red-shifted due to an increase of intermolecular interactions. Strongly red-shifted emission spectra due to intermolecular vibronic coupling are observed for films and single crystals of p,p'-dicyano-distyrylbenzene with pi,pi-stacking of the molecules.

The diffusion coefficients, D, of oxygen in titaniumoxo phthalocyanine (TiOPc) films are determined by following the increase of electric photoconductivity of the films upon oxygen sorption. The diffusion coefficient in as-deposited films of unsubstituted TiOPc is D approximate to 8 x 10(-16) cm(2) s(-1). Introduction of alkyl spacers leads to the increase of D by an order of magnitude. Scanning electron micrographs and extended X-ray absorption fine structure (EXAFS) measurements show that this increase is due to the amorphous structure and the larger distances between the conjugated pi-systems in films of substituted Pc's. As photo-oxidation of the films is a diffusion controlled process, its quantum yield. phi(p). increases 10-fold upon alkyl-substitution.Annealing the films leads to microcrystalline films with a closer alignment of the pi-conjugated systems, as demonstrated by the changes in the UV-Vis absorption spectra. Consequently, D decreases by approximately an order of magnitude. (C) 2003 Elsevier Science B.V. All rights reserved.