Applied Spectroscopy

Several methodologies have been developed over the past several years for super-resolution fluorescence microscopy including saturated structured-illumination microscopy (SSIM), stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). While they have shown great promise for biological research, these techniques all have individual strengths and weaknesses. This review will describe the basic principles for achieving super resolution, demonstrate some applications in biology, and provide an overview of technical considerations for implementing these methods.

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.

Light harvesting and energy transfer between allophycocyanin (APC) and a tandem conjugate dye was investigated using single-molecule recrossings in the probe volume. By comparing saturation irradiance and recrossing events of APC and the tandem conjugate Streptavidin Allophycocyanin- AlexaFluor-680 (APC-680), it was demonstrated that the light tolerance and photostability of APC-680 were improved over APC. This improvement was due to conjugating a tandem dye to APC, which provided an alternative pathway for energy transfer. This additional pathway decreases photobleaching and triplet-state conversion. In this study, the average fluorescence intensity at different laser powers was calculated from extracted signal peaks. Saturation irradiance was determined by interpolating the average intensity as a function of excitation power. By comparing the saturation irradiance of APC and APC-680, it was demonstrated that light tolerance was not significantly increased by having a small fluorophore covalently linked to the protein. The molecular recrossing method was used to determine triplet-state conversion and photobleaching effects. The number of molecules that were detected within 2 ms of each other was defined as the number of recrossing events (Nr). The total number of molecules detected was defined as N_t. The normalized number of recrossings events (N_r/N_t) showed that energy transfer between APC and AlexaFluor-680 could reduce triplet-state formation of phycobiliproteins. In this case, the saturation irradiance was not improved, but the resistance to photobleaching and triplet-state conversion was improved.

We report the fluorescence and surface-enhanced Raman scattering (SERS) analysis of methyl red (MR) isomers (o-MR, m-MR, p-MR) on silver (Ag) colloids at different pH conditions. The changes in simultaneous fluorescence quenching around 400 nm accompanied by enhancement around 540 nm with pH are closely related to energy transfer efficiency and the interaction between the carboxylic group and the Ag surface. The change of SERS signals with pH values can be ascribed to different adsorption orientations of MR on the Ag surface, from near flat (o-MR) to near perpendicular (p-MR).

Eighty drugs of abuse and metabolites were successfully measured by surface-enhanced Raman spectroscopy (SERS) using gold- and silver-doped sol-gels immobilized in glass capillaries. A method was developed that provided consistent detection of 50 ppb cocaine in saliva in a focused study. This general method was successfully applied to the detection of a number of additional drugs in saliva, such as amphetamine, diazepam, and methadone.

Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation, the mechanisms of biological niche creation, and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However, the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supra-cellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e., 5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios, a differentiation status indicator observed previously using Raman spectroscopy, confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition, pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations, which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed, with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases, cell density, pluripotency, and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 μm and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches.

Attenuated total reflection (ATR) infrared and Fourier transform (FT) Raman spectra were obtained from wool threads dyed in the laboratory with natural dyes used in antiquity, following a procedure similar to ancient methods for dyeing wool. The ATR spectra were primarily dominated by the signals of the wool, making it difficult to identify the dye on the fibers only by visual inspection of the infrared spectrum. However, the Raman spectra showed more significant characteristics attributable to the dyes as previously studied in the literature on modern synthetic dyes. A library-search method was thus applied to the second derivatives of both the ATR and Raman spectra to verify the possibility of identifying the dye. Two libraries were constructed, one consisting of the ATR spectra of undyed wool (raw, washed, and mordanted) and the transmission spectra of pure dyes and the other consisting of the Raman spectra of undyed wool and of pure dyes. Correlation and first-derivative correlation search algorithms were used. The results presented here suggest that the two types of spectroscopy are complementary in this kind of work, allowing the almost complete identification of historic dyes on wool. In fact, through the combined use of the two searches, most dyes were identified with a good index of similarity and within the first five hits. Only for annatto was identification totally impossible using either technique. Subsequently the same method was applied to wool, silk, and cotton threads taken from ancient Caucasian and Chinese textiles.

Water-in-oil (w/o) emulsions are of great interest in many areas including food technology and the oil and gas industry. However, the molecular mechanisms that lead to a stable emulsion are yet to be fully understood. In this article, the potential of attenuated total reflection (ATR) infrared (IR) spectroscopy for studying the influence of an emulsifier on the molecular water structure in a thin layer at the w/o interface is demonstrated. For this purpose, IR spectra from a bilberry extract w/o emulsion are analyzed. The thickness of the probed water layer is estimated to be below 0.5 μm, which is well below the droplet diameter. The IR spectra recorded in aqueous solution and the w/o emulsion reveal a strengthening of the intramolecular covalent O-H bonds in the presence of the emulsifier, which in turn indicates a change in the hydrogen bond network in terms of weakening the intermolecular interactions in the water layer at the interface.

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319–3228 (2001) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its M_F and M_S components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm⁻¹ for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the M_F pathway of the parallel cycles model of the photocycle.

Attenuated total reflection (ATR) infrared absorption spectroscopy is a well-known vibrational spectroscopy technique for many different applications. In recent years this technique has been used to detect thin layer(s) lying on a solid substrate. Such a sample needs high pressure to ensure good optical contact between sample and prism and a p-polarization to enhance the signal to be detected. Such conditions have not been detailed in the literature regarding the effect of high pressure on the ATR measurement. This study shows the detrimental effect of high pressure on the ATR spectra. This effect is related to light depolarization induced by the germanium prism under high pressure. Moreover, the importance of polarizer position in the optical bench is highlighted. Indeed, due to the pressure-induced depolarization of the prism, the polarizer has to be placed before the prism to limit undesirable effects on the ATR spectrum baseline.

Time-resolved Fourier transform near-infrared (FT-NIR) spectroscopic imaging was applied to the diffusion process of butanol(OD) into polyamide 11 (PA11) with a novel sheet-structured variable-temperature-controlled sample holder in order to demonstrate the significant differences of diffusion rate below and above the glass transition temperature of PA11. The diffusant butanol(OD) was chosen for two reasons: (1) it allows the diffusion front to be monitored by the intensity decrease of a NH-specific absorption band of PA11 due to the NH/ND isotope exchange and (2) under the measurement conditions the diffusion of butanol(OD) into PA11 takes place in an experimentally manageable time frame. Apart from the in situ visualization of the diffusion front in the time-resolved FT-NIR images, the type of diffusion and the diffusion coefficient of butanol(OD) into PA11 have been determined.

Visible and near-infrared (Vis-NIR, 350–2500 nm) diffuse reflection spectroscopy (DRS) models built from “as-collected” samples of solid cattle manure accurately predict concentrations of moisture and crude ash. Because different organic molecules emit different spectral signatures, variations in livestock diet composition may affect the predictive accuracy of these models. This study investigates how differences in livestock diet composition affect Vis-NIR DRS prediction of moisture and crude ash. Spectral signatures of solid manure samples (n = 216) from eighteen groups of cattle on six different diets were used to calibrate and validate partial least squares (PLS) regression models. Seven groups of PLS models were created and validated. In the first group, two-thirds of all samples were randomly selected as the calibration set and the remaining one-third were used for the validation set. In the remaining six groups, samples were grouped by livestock diet (ration). Each ration in turn was held out of calibrations and then used as a validation set. When predicting crude ash, the fully random calibration model produced a root mean square deviation (RMSD) of 2.5% on a dry basis (db), ratio of standard error of prediction to the root mean squared deviation (RPD) of 3.1, bias of 0.14% (db), and correlation coefficient r² of 0.90., When predicting moisture, an RMSD of 1.5% on a wet basis (wb), RPD of 4.3, bias of −0.09% (wb), and r² of 0.95 was achieved. Model accuracy and precision were not impaired by exclusion of any single ration from model calibration.

Epigallocatechin-3-gallate (EGCG) is credited with the majority of the health benefits associated with green tea consumption. It has a high economic and medicinal value. The feasibility of using different variable selection approaches in Fourier transform near-infrared (FT-NIR) spectroscopy for a rapid and conclusive quantitative determination of EGCG in green tea was investigated. Graphically oriented multivariate calibration modeling procedures such as interval partial least squares (iPLS), synergy interval partial least squares (siPLS), and genetic algorithm optimization combined with siPLS (siPLS-GA) were applied to select the most efficient spectral variables that provided the lowest prediction error. The performance of the final model was evaluated according to the root mean square error of prediction (RMSEP) and coefficient of determination (R²) for the prediction set. Experimental results showed that the siPLS-GA model obtained the best results in comparison to other models. The optimal models were achieved with R²_p = 0.97 and RMSEP=0.32. The model can be obtained with only 36 variables retained and it provides a robust model with good estimation accuracy. This demonstrates the potential of NIR spectroscopy with multivariate calibration methods to quickly detect the bioactive component in green tea.

Self-assembly and disassembly behavior of a series of chiral sorbitol-based derivatives in n-octanol were investigated by using the variable temperature circular dichroism (CD) technique. An attempt has been made to establish a novel strategy for the characterization of the self-assembly/disassembly transition by measuring the difference of CD signals of chiral aggregates.

This paper presents a developed dielectric-barrier-discharge-based “sniffer” that offers unique characteristics not available from other techniques. It is a portable, highly specific, and sensitive detector that operates at atmospheric pressure. It provides both molecular and elemental information on organic and inorganic gases and particulate aerosols. Measurements were made to electrically characterize the plasma and calculate the energy coupled into the plasma. We created a signature database for diverse chemicals based on the atomic and diatomic emission spectrum that serves to classify the compound and ideally recognize it by composition with the optical emission intensity corresponding to concentration. For some operational regimes and species, emission from OH (A²Σ⁺-X²π), CH (A²Δ-X²π), and often C₂ (d³π_g-a³π_u; Swan band system) diatomic radicals is produced. Limits of detection extend to parts per billion (ppb) levels for some species such as decane, 2-decanol, and nitrobenzene. Results are presented for differentiation of classes of organic compounds such as alkanes, aromatics, oxygenates, chlorinated, and nitrogen-containing organic compounds.

A portable spectrometer prototype is built to study wavelength calibration for flat-field grating spectrometers. An accurate calibration method called parameter fitting is presented. Both optical and structural parameters of the spectrometer are included in the wavelength calibration model, which accurately describes the relationship between wavelength and pixel position. Along with higher calibration accuracy, the proposed calibration method can provide information about errors in the installation of the optical components, which will be helpful for spectrometer alignment.