Spectral Reproducibility Research Articles

Impact of fixation on in vitro cell culture lines monitored with Raman spectroscopy

Raman spectroscopy provides chemical-rich information about the composition of analytes and is a powerful tool for biological studies. With the ability to investigate specific cellular components or image whole cells, compatible methods of sample preservation must be implemented for accurate spectra to be collected. Unfortunately, the effects of many commonly used sample preservation methods have not been explored with cultured cells. In this study, two human cell lineages of varying phenotypes were used to investigate the effects of sample preservation methods. Cells were cultured directly onto quartz substrates and either formalin-fixed, desiccated or air dried. The results indicate that the methodology applied to cell cultures for Raman analysis significantly influences the quality and reproducibility of the resulting spectral data. Formalin fixation was not found to be as universally efficient as anticipated for a commonly used fixative. This was due largely to the inconsistency in sample preservation between cell lines and loss of signal intensity. Sample air-drying was found to be largely inconsistent in terms of spectral reproducibility. Our study shows that sample desiccation displayed good spectral reproducibility and resulted in a good signal-to-noise ratio. Lipid and protein content in both activated and inactivated cells were maintained and provided a more controlled method compared with air-drying, revealing that the speed of drying is important for sample preservation.

A novel approach to correct variations in Raman spectra due to photo-bleachable cellular components

Bacterial typing by Raman spectroscopy is based on small spectral differences that exist between strains, due to differences in their overall molecular composition. These strain-specific spectral differences can be obscured by sources of non-specific signal variance. One such source is the signal contribution of microbial pigments that can vary strongly in intensity. Examples of such pigments are carotenoids in Staphylococcus aureus, and other pigments in Pseudomonas aeruginosa and Mycobacterium lentiflavum. The variance in the intensity of these pigments greatly overshadows strain-specific differences, and therefore lowers spectral reproducibility and causes misclassification of microbial strains. Here a method is presented to determine the spectral signature of pigments of which the relative signal contribution decreases under laser irradiation; so-called photo-bleachable pigments. These signatures are used to eliminate signal variance caused by these pigments by means of the extended multiplicative scatter correction algorithm and spectral interferent subtraction. Application of this method increases the reproducibility of the spectra of microorganisms that contain such pigments to the extent that reproducible identification of samples at strain level is achieved.

MALDI-TOF mass signatures for differentiation of yeast species, strain grouping and monitoring of morphogenesis markers

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is demonstrated to be a potentially useful tool for the rapid identification of yeasts, the grouping of Candida albicans strains, and the monitoring of germ tube-specific markers. Co-crystallized with sinapinic acid as the MALDI matrix, intact yeast cells yielded a sufficient number of medium-sized ions (4-15 kDa) in MALDI mass spectra to provide "mass signatures" that were diagnostic of strain type. For most isolates, the mass signatures were affected by the growth medium, length of incubation and the cell preparation method. While the overall past success of this methodology for fungal cells has been relatively low compared to its application to bacteria, fixing the yeast cells in 50% methanol inactivated the cells, reduced cell aggregation in aqueous suspension solution, and more importantly, it significantly improved the mass signature quality. This simple but critical advance in sample treatment improved mass spectrometric signal-to-noise ratios and allowed the identification of yeasts by a mass signature approach. Under optimized conditions, Candida species (C. albicans, C. glabrata, C. krusei, C. kefyr), Aspergillus species (A. terreus, A. fumigatus, A. syndowii) and other yeast genera (Cryptococcus neoformans, Saccharomyces cerevisiae and a Rhodotorula sp.) could be distinguished. Within the C. albicans species, several common ions in the m/z 5,000-10,000 range were apparent in the mass spectra of all tested strains. In addition to shared ions, the mass spectra of individual C. albicans strains permitted grouping of the strains. Principal component analysis (PCA) was employed to confirm spectral reproducibility and C. albicans strain grouping by mass signatures. Finally, C. albicans germ tubes produced MALDI-TOF mass signatures that differed from yeast forms of this species. This is a rapid, sensitive and simple method for identifying yeasts, grouping strains and following the morphogenesis of C. albicans.

NMR observation of Tau in Xenopus oocytes

The observation by NMR spectroscopy of microinjected 15N-labelled proteins into Xenopus laevis oocytes might open the way to link structural and cellular biology. We show here that embedding the oocytes into a 20% Ficoll solution maintains their structural integrity over extended periods of time, allowing for the detection of nearly physiological protein concentrations. We use these novel conditions to study the neuronal Tau protein inside the oocytes. Spectral reproducibility and careful comparison of the spectra of Tau before and after cell homogenization is presented. When injecting Tau protein into immature oocytes, we show that both its microtubule association and different phosphorylation events can be detected.

Highly Efficient Classification and Identification of Human Pathogenic Bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

Effects of the application of different window functions and projection methods on processing of 1H J-resolved nuclear magnetic resonance spectra for metabolomics

Two dimensional (2D) homonuclear 1H J-resolved (JRES) nuclear magnetic resonance spectroscopy is increasingly used in metabolomics. This approach visualises metabolite chemical shifts and scalar couplings along different spectral dimensions, thereby increasing peak dispersion and facilitating spectral assignments and accurate quantification. Here, we optimise the processing of 2D JRES spectra by evaluating different window functions, a traditional sine-bell (SINE) and a combined sine-bell–exponential (SEM) function. Furthermore, we evaluate different projection methods for generating 1D projected spectra (pJRES). Spectra were recorded from three disparate types of biological samples and evaluated in terms of sensitivity, reproducibility and resolution. Overall, the SEM window function yielded considerably higher sensitivity and comparable spectral reproducibility and resolution compared to SINE, for both 1D pJRES and 2D JRES datasets. Furthermore, for pJRES spectra, the highest spectral quality was obtained using SEM combined with skyline projection. These improvements lend further support to utilising 2D J-resolved spectroscopy in metabolomics.

Q-BAND EPR BIODOSIMETRY IN TOOTH ENAMEL MICROSAMPLES: FEASIBILITY TEST AND COMPARISON WITH X-BAND

A comparative study of electron paramagnetic resonance dosimetry in Q- and X-bands has shown that Q-band is able to provide accurate measurements of radiation doses even below 0.5 Gy with tooth enamel samples as small as 2 mg. The optimal amount of tooth enamel for dose measurements in Q-band was found to be 4 mg. This is less than 1% of the total amount of tooth enamel in one molar tooth. Such a small amount of tooth enamel can be harmlessly obtained in an emergency requiring after-the-fact radiation dose measurement. The other important advantage of Q-band is full resolution of the radiation-induced EPR signal from the native, background signal. This separation makes dose response measurements much easier in comparison to conventional X-band measurements in which these overlapping signals necessitate special methods for doses below 0.5 Gy. The main disadvantages of Q-band measurements are a higher level of noise and lower spectral reproducibility than in X-band. The effect of these negative factors on the precision of dose measurements in Q-band could probably be reduced by improvement of sample fixation in the resonance cavity and better optimization of signal filtration to reduce high-frequency noise.

Evaluating Reproducibility and Similarity of Mass and Intensity Data in Complex Spectra—Applications to Tubulin

We present a data processing approach based on the spectral dot product for evaluating spectral similarity and reproducibility. The method introduces 95% confidence intervals on the spectral dot product to evaluate the strength of spectral correlation; it is the only calculation described to date that accounts for both the non-normal sampling distribution of the dot product and the number of peaks the spectra have in common. These measures of spectral similarity allow for the recursive generation of a consensus spectrum, which incorporates the most consistent features from statistically similar replicate spectra. Taking the spectral dot product and 95% confidence intervals between consensus spectra from different samples yields the similarity between these samples. Applying the data analysis scheme to replicates of brain tubulin CNBr peptides enables a robust comparison of tubulin isotype expression and post-translational modification patterns in rat and cow brains.

Metabolic Imaging of Myocardial Triglyceride Content: Reproducibility of1H MR Spectroscopy with Respiratory Navigator Gating in Volunteers

Institutional review board approval and informed consent were obtained. The purpose of the study was to prospectively compare spectral resolution and reproducibility of hydrogen 1 (1H) magnetic resonance (MR) spectroscopy, with and without respiratory motion compensation based on navigator echoes, in the assessment of myocardial triglyceride content in the human heart. In 20 volunteers (14 men, six women; mean age+/-standard error, 31 years+/-2.8 [range, 19-60 years]; body mass index, 19-30 kg/m2) without history of cardiovascular disease, 1H MR spectroscopy of the myocardium was performed at rest, with and without respiratory motion compensation. Unsuppressed water signal linewidth changed from 11.9 Hz to 10.7 Hz (P<.001) with the use of the navigator, which indicated better spectral resolution. The navigator improved the intraclass correlation coefficient for the assessment of myocardial triglyceride content from 0.32 to 0.81. Therefore, the authors believe that respiratory motion correction is essential for reproducible assessment of myocardial triglycerides.

A new non-invasive, quantitative Raman technique for the determination of an active ingredient in pharmaceutical liquids by direct measurement through a plastic bottle

The concentration of an active pharmaceutical ingredient (povidone) in a commercial eyewash solution has been measured directly through a plastic (low-density polyethylene: LDPE) container using a wide area illumination (WAI) Raman scheme. The WAI scheme allows excitation using a 6 mm laser spot (focal length: 248 mm) that is designed to cover a wide sample area. As a result, it has the potential to improve the reliability Raman measurements by significantly enhancing representative sample interrogation, thus improving the reproducibility of sampling. It also decreases the sensitivity of sample placement with regard to the excitation focal plane. Simultaneously, isobutyric anhydride was placed in front of the bottles to use for a synchronous external standard configuration. This helps to correct the problematic variation of Raman intensity from the inherent fluctuation in laser power. Using the WAI Raman scheme combined with the synchronous standard method, the povidone concentration was successfully measured with spectral collection that was performed through a plastic barrier. The conventional Raman scheme was difficult to employ for the same purpose because of the degraded spectral reproducibility resulting from the smaller laser illumination area and the sensitivity of such an approach to the position of the sample bottle. The result from this study suggests that the WAI scheme exhibits a strong potential for the non-destructive quantitative analysis of pharmaceuticals measured directly in plastic containers. Preliminary work also shows that similar measurements can also be made in glass bottles. If implemented, this technique could be utilized as a simple and rugged method for quality assurance of final products in a manner consistent with Process analytical technology (PAT) requirements.

Raman-based detection of bacteria using silver nanoparticles conjugated with antibodies

Surface enhanced Raman scattering (SERS) has been used to detect bacteria captured by polyclonal antibodies sorbed onto protein-A-modified silver nanoparticles. The selectivity and discrimination of the technique were assured by using a specific antibody to the model bacterium, Escherichia coli. As the SERS enhancement mechanism depends upon the metal surface proximity, 8 nm was considered as the optimum distance between the bacterium and the nanoparticle surface. Spectral reproducibility was verified using Principal Components Analysis to differentiate the clusters corresponding to the biomolecules and/or bacteria sorbed onto nanoparticles. Compared to the normal Raman spectrum, the SERS technique resulted in an intensity enhancement of over 20-fold.

Multispectral Image Compression for Improvement of Colorimetric and Spectral Reproducibility by Nonlinear Spectral Transform

The article proposes a multispectral image compression scheme using nonlinear spectral transform for better colorimetric and spectral reproducibility. In the method, we show the reduction of colorimetric error under a defined viewing illuminant and also that spectral accuracy can be improved simultaneously using a nonlinear spectral transform called Labplus, which takes into account the nonlinearity of human color vision. Moreover, we show that the addition of diagonal matrices to Labplus can further preserve the spectral accuracy and has a generalized effect of improving the colorimetric accuracy under other viewing illuminants than the defined one. Finally, we discuss the usage of the first-order Markov model to form the analysis vectors for the higher order channels in Labplus to reduce the computational complexity. We implement a multispectral image compression system that integrates Labplus with JPEG2000 for high colorimetric and spectral reproducibility. Experimental results for a 16-band multispectral image show the effectiveness of the proposed scheme.

MALDI Tissue Imaging of Ocular Lens α-Crystallin

To apply MALDI (matrix-assisted laser desorption ionization) tissue imaging methods to obtaining a profile of the distribution of the lens alpha-crystallins and their modified forms in calf and mature bovine lenses. Frozen bovine lenses were cut equatorially at -12 degrees C to -20 degrees C into 10- to 40-microm sections depending on lens age. Tissue sections were mounted onto MALDI sample plates by ethanol soft-landing to maintain tissue integrity. A two-layered matrix deposition method was used to improve mass spectral reproducibility across sections. Molecular images of the two subunits of alpha-crystallin and their modifications over approximately one-half of a single tissue section were reconstituted from mass spectral data sets acquired in 250-microm steps. Identification of protein truncation products and confirmation of phosphorylation distribution patterns were performed by reverse-phase liquid chromatography of soluble extracts from specific tissue regions followed by tandem mass spectrometry (LC/MS/MS). Distinct distribution patterns were observed for the two subunits of alpha-crystallin and their modified forms. alphaA-crystallin showed extensive truncation across whole sections, especially in the nuclei, whereas alphaB-crystallin was observed to be relatively stable. Both alphaA-crystallin and alphaB-crystallin displayed the highest level of phosphorylation in the middle cortex region, a finding confirmed by LC/MS/MS analysis of dissected regions. A new imaging technique has been successfully applied to molecularly characterize the spatial distribution of lens proteins and their modifications in lens sections. The different distributions of alpha-crystallin revealed in this study provide new leads in the investigation of underlying physiological significance of the modified forms of the two alpha-crystallin subunits.

In vivo31P MR spectral patterns and reproducibility in cancer patients studied in a multi‐institutional trial

The standardization and reproducibility of techniques required to acquire anatomically localized 31P MR spectra non-invasively while studying tumors in cancer patients in a multi-institutional group at 1.5 T are reported. This initial group of patients was studied from 1995 to 2000 to test the feasibility of acquiring in vivo localized 31P MRS in clinical MR spectrometers. The cancers tested were non-Hodgkin's lymphomas, sarcomas of soft tissue and bone, breast carcinomas and head and neck carcinomas. The best accrual and spectral quality were achieved with the non-Hodgkin's lymphomas. The initial analysis of the spectral values of the sum of phosphoethanolamine plus phosphocholine normalized by the content of nucleotide triphosphates in a homogeneous sample of 32 NHL patients studied by in vivo (31)P MRS showed good reproducibility among different institutions. No statistical differences were found between the institution with the largest number of cases accrued and the rest of the multi-institutional NHL data (2.28 +/- 0.64, mean +/- standard error; n = 17, vs 2.08 +/- 0.14, n = 15). The preliminary data reported demonstrate that the institutions involved in this trial are obtaining reproducible 31P MR spectroscopic data non-invasively from human tumors. This is a fundamental prerequisite for the international cooperative group to be able to demonstrate the clinical value of the normalized determination of phosphoethanolamine plus phosphocholine by 31P MRS as predictor for treatment response in cancer patients.

Multispectral Image Compression for High Fidelity Colorimetric and Spectral Reproduction

The article aims to provide a solution for multispectral image compression for high color reproducibility with preservation to spectral accuracy. In the method previously proposed to reduce the colorimetric error of the reconstructed multispectral image, a weighting matrix is incorporated to Karhunen-Loeve transform (KLT) as the spectral transform for multispectral image compression, which accounts for the color matching functions of human observers as well as the viewing illuminants. However, the colorimetric improvements are obtained on the cost of degradation of spectral accuracy. In this paper, we show that the reduction of colorimetric error and the preservation of spectral accuracy is a tradeoff that can be controlled by adding a diagonal matrix that is composed of a scalar multiple of an identity matrix to the weighting matrix of KLT. As the result, the small values in the weighting matrix can be lifted up, thus reduce the spectral errors in the corresponding reconstructed multispectral image bands. We implement a multispectral image compression system that integrates the proposed spectral transforms with the addition of diagonal matrix and JPEG2000 for high colorimetric and spectral reproducibility. Experimental results for three 16-band multispectral images show that spectral accuracy can be improved without loss of substantial color reproducibility if the magnitude of the scalar in the diagonal matrix is chosen appropriately

Automated Acoustic Matrix Deposition for MALDI Sample Preparation

Novel high-throughput sample preparation strategies for MALDI imaging mass spectrometry (IMS) and profiling are presented. An acoustic reagent multispotter was developed to provide improved reproducibility for depositing matrix onto a sample surface, for example, such as a tissue section. The unique design of the acoustic droplet ejector and its optimization for depositing matrix solution are discussed. Since it does not contain a capillary or nozzle for fluid ejection, issues with clogging of these orifices are avoided. Automated matrix deposition provides better control of conditions affecting protein extraction and matrix crystallization with the ability to deposit matrix accurately onto small surface features. For tissue sections, matrix spots of 180-200 microm in diameter were obtained and a procedure is described for generating coordinate files readable by a mass spectrometer to permit automated profile acquisition. Mass spectral quality and reproducibility was found to be better than that obtained with manual pipet spotting. The instrument can also deposit matrix spots in a dense array pattern so that, after analysis in a mass spectrometer, two-dimensional ion images may be constructed. Example ion images from a mouse brain are presented.

Electrospray interfacing of polymer microfluidics to MALDI-MS

The off-line coupling of polymer microfluidics to MALDI-MS is presented using electrospray deposition. Using polycarbonate microfluidic chips with integrated hydrophobic membrane electrospray tips, peptides and proteins are deposited onto a stainless steel target followed by MALDI-MS analysis. Microchip electrospray deposition is found to yield excellent spatial control and homogeneity of deposited peptide spots, and significantly improved MALDI-MS spectral reproducibility compared to traditional target preparation methods. A detection limit of 3.5 fmol is demonstrated for angiotensin. Furthermore, multiple electrospray tips on a single chip provide the ability to simultaneously elute parallel sample streams onto a MALDI target for high-throughput multiplexed analysis. Using a three-element electrospray tip array with 150 microm spacing, the simultaneous deposition of bradykinin, fibrinopeptide, and angiotensin is achieved with no cross talk between deposited samples. In addition, in-line proteolytic digestion of intact proteins is successfully achieved during the electrospray process by binding trypsin within the electrospray membrane, eliminating the need for on-probe digestion prior to MALDI-MS. The technology offers promise for a range of microfluidic platforms designed for high-throughput multiplexed proteomic analyses in which simultaneous on-chip separations require an effective interface to MS.

Surface-Enhanced Micro-Raman Detection and Characterization of Calix[4]Arene—Polycyclic Aromatic Hydrocarbon Host—Guest Complexes

Surface-enhanced micro-Raman spectroscopy (micro-SERS) was used to detect traces of the hazardous pollutant polycyclic aromatic hydrocarbons (PAHs) pyrene and benzo[c]phenanthrene deposited onto a calix[4]arene-functionalized Ag colloidal surface. High spectral reproducibility and very low molecular detection limits (10(-8) M) were obtained by using 25,27-carboethoxy-26,28-hidroxy-p-tert-butylcalix[4]arene as host molecule. Films of immobilized aggregated Ag nanoparticles, obtained by chemical reduction with hydroxylamine, were prepared by direct adhesion on a glass surface. The influence of the aggregation degree of the initial Ag nanoparticles on the micro-SERS detection effectiveness was checked. Different relative concentrations of the host (calixarene receptor) and the guest (PAHs) were attempted in order to optimize detection of the pollutant. The obtained results indicated that the detection limit is much lower in the case of benzo[c]phenanthrene than in pyrene when exciting with the 785 nm line of a diode laser. A detailed interpretation of the Raman spectra was accomplished in order to obtain more information about the interaction mechanism of the host-guest complex, which could be useful in the future for the design of powerful detection systems.

Development of a PLS based method for determination of the quality of beers by use of NIR: spectral ranges and sample-introduction considerations

Near infrared spectroscopy (NIR) has been used to determine important indicators of the quality of beers, for example original and real extract and alcohol content, using a partial least squares (PLS) calibration approach. A population of 43 samples, obtained commercially in Spain and including different types of beer, was used. Cluster hierarchical analysis was used to select calibration and validation data sets. Absorbance sample spectra, in transmission mode, were obtained in triplicate by using a 1-mm pathlength quartz flow cell and glass chromatography vials of 6.5 mm internal diameter. The two methods of sample introduction were compared critically, on the basis of spectral reproducibility for triplicate measurements and after careful selection of the best spectral pre-processing and the spectral range for building the PLS model, to obtain the best predictive capability. For each mode of sample introduction two calibration sets were assayed, one based on the use of 15 samples and a second extended based on use of 30 samples, thus leaving 28 and 13 samples, respectively, for validation. The best results were obtained for 1 mm flow cell measurements. For this method original zero-order spectra data in the ranges 2220-2221 and 2250-2350 nm were chosen. For the real extract, original extract, and alcohol d(x-y) and s(x-y) values of -0.04 and 0.07% w/w, -0.01 and 0.13% w/w, and -0.01 and 0.1% v/v, respectively, were obtained. The maximum errors in the prediction of any of these three indicators for a new sample were 2.2, 1.2, and 1.9%, respectively. This method compares favorably with the automatic reference method in terms of speed, reagent consumed, and waste generated.

Static TOF-SIMS — a VAMAS interlaboratory study. Part I. Repeatability and reproducibility of spectra

An interlaboratory study involving 32 Time-of-Flight Static SIMS instruments from 12 countries has been conducted. Analysts were supplied, by NPL, with a protocol for analysis together with three reference materials; a thin layer of polycarbonate (PC) on a silicon wafer, a thin layer of polystyrene (PS) oligomers on etched silver and poly(tetrafluoroethylene) (PTFE). The study involved static SIMS analysis of each reference material for both positive and negative polarity secondary ions. The option to test instrument suitability for G-SIMS was also provided. The results of this study show that over 84% of instruments have excellent repeatabilities of better than 1.9%. Repeatabilities can be as good as 0.4%. A relative instrument spectral response (RISR) is calculated for each instrument for each reference material and ion polarity. The RISR is used to evaluate variations in spectral response between different generic types of SIMS instruments. Use of the RISR allows the identification of contamination, charge stabilisation problems and incorrectly functioning ion detectors. The high quality of the data presented here allows the RISR to reveal differences in individual operation of each instrument such as the use of apertures to remove metastables from the spectra and the use of different post-acceleration voltages for ion detection. Spectral reproducibility can be measured, here, by the equivalence of RISRs between materials and ion polarities. It is found that reproducibilities are on average 10% but can be as good as 4% for the best instruments. This figure shows the consistency between instruments in measuring spectra from different samples. This study sets out the basic framework to develop static secondary ion mass spectrometry (SSIMS) as a reliable measurement method. © Crown Copyright 2005. Reproduced with the permission of Her Majestry's Stationery Office. Published by John Wiley & Sons, Ltd.