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We report on the reflectance, transmittance and fluorescence spectra (λ=200–1200nm) 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 color pigments 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 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.
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.
