Spectral decomposition of intracellular complex fluorescence using multiple-wavelength phase modulation lifetime determination: technical approach and preliminary applications

Lifetime-based spectral decomposition using a frequency-domain phase/modulation technique is developed on a microspectrofluorimeter prototype. In a fluorescent mixture with strongly overlapping components, such measurements enable us to not only obtain excited state lifetimes of each fluorescent component but also determine the specific spectral contribution of each species without the use of any model spectra. Examples of such applications are first given for complex mixtures of highly overlapping fluorescent components in solution. Preliminary results concerning cellular applications are also reported. This allows us to follow the cellular uptake and intracellular stability of fluorescent labeled modified oligonucleotides in the context of antisense strategy studies. Indeed, the intracellular signal from the fluorescent label bound to oligonucleotides can be distinguished from those of the free label by its specific excited state lifetime.     

A computer analysis of electrophoresis and ultracentrifugation patterns.

A program DIANA is designed to fit the scanning curves of electrophoresis and ultracentrifugation patterns with 10-15 overlapping peaks by a sum of Gaussian-like distribitions. The parameters of the distributions are adjusted by minimizing x, using a problem oriented minimizing procedure. The number of molecular components as well as starting values of the positions and the widths of the peaks must be choosen as input for each type of pattern. Programs are written in FORTRAN IV.     

Blind spectral decomposition of single-cell fluorescence by parallel factor analysis

Simultaneous measurement of multiple signaling molecules is essential to investigate their relations and interactions in living cells. Although a wide variety of fluorescent probes are currently available, the number of probes that can be applied simultaneously is often limited by the overlaps among their fluorescence spectra. We developed the experimental system to measure and analyze many overlapping fluorescent components in single cells. It is based on the recording of two-dimensional single-cell fluorescence spectra and on the blind spectral decomposition of fluorescence data by method of parallel factor analysis. Because this method does not require any preknowledge about the shapes of individual component spectra, it can be applied to the specimens that contain fluorescent components with unknown spectra. By examining the performance using the mixture solutions of fluorescent indicators, it was confirmed that >10 largely overlapping spectral components could be easily separated. The effectiveness in the physiological experiments was proven in the applications to the temporal analysis of intracellular Ca(2+) concentration and pH, as well as the intrinsic fluorescent components, in single mouse oocytes.     

The nature of very low frequency component of the heart rate variability and the role of sympathetic-parasympathetic interactions

A method of separate monitoring "instant" changes of the VLF, LF and HF power spectral components of heart rate variability, has been developed. The power of the LF and HF spectral components were proved to be continuously changing. The period of these power fluctuations could stay within 15 to 150 sec. Comparison of the heart rate variability spectrum with LF and HF spectral components power fluctuations' spectrums has shown that the frequencies of the LF and HF spectral components power fluctuations stay within the VLF range. The co-operative spectrum form of these fluctuations repeats the form of the VLF peak. In cases when the LF and HF spectral components power fluctuations' periods do not coincide, VLF has two peaks. The frequency of one VLF peak coincides with frequency of the HF power fluctuations, and the frequency of another--with the frequency of LF power fluctuations.     

Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra

Two procedures for analyzing overlapping optical spectra of mixtures of pyridine hemochromes are described, and extinction coefficients of pyridine hemochromes are provided for use with these methods. In the first procedure, absorbance is measured at a number of wavelengths equal to the number of components to be analyzed. This is the minimum amount of spectral data from which the concentration of each species can be calculated. In the second procedure, absorbance is measured at a number of wavelengths greater than the number of components to be analyzed. This redundancy of information makes it impossible to fit spectra which contain contributions from additional components, unless the spectra of the additional components are equal to linear combinations of the spectra of the species being analyzed. These two procedures are generally applicable to analyses of absolute or difference spectra of mixtures of components obeying Beer's law. The sensitivity to error in the absorbance measurements is only slightly greater than that for measuring a pure component at a single wavelength.     

Decomposition fluorescence spectra of tryptophan residues in proteins based on log-normal components by a least squares method]

An algorithm of decomposition of protein tryptophan spectra into components was developed. The spectral shape of components is described by a uniparametric log-normal function. Rise of certainty and accuracy of resolution of widely overlapping smooth spectral components (a typical uncorrect reverse problem) was achieved using several regularizing factors: (i) the set of experimental spectra used were measured at several quencher concentrations; (ii) the functional being minimized, along with the root mean square residuals of intensities, the term depending on the obedience to the Stern-Volmer law; (iii) an extra information is used--the number of experimental values greatly exceeds the number of parameters to be estimated. The minimum of functional is determined by a consecutive setting of all possible combinations of component spectral maxima values, which allows to avoid sticking in the local minima of noisy functional. The real experimental noise restricts the decomposition into not more than three components. The decomposition error does not exceed the experimental one. The algorithm functioning is illustrated by resolution of tryptophan fluorescence spectra of papain into one, two, and three components.     

The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed.

Spectral imaging is a natural extension of the capabilities of confocal microscopes. The first confocal spectral imaging (CSI) instruments were able to acquire spectral data that allowed the emissions of overlapping fluorescent probes to be assigned to data channels representing a spectrum rather than a range of emission wavelengths. This marked a significant improvement over what could be done by channel series with standard confocal microscopes. However the performance of these earlier designs can fall short in one or more of the following areas; sensitivity, spectral resolution and reproducibility, acquisition speed, and unmixing accuracy. Nikon has recently introduced a new CSI instrument, C1si, that overcomes some of the more serious performance deficiencies of earlier designs through unique optical, electronic, and data handling advances. C1si uses a multianode photomultiplier tube (PMT) as the detector and typically acquires spectral data in a single scan. Sensitivity is enhanced over designs diffracting randomly polarized fluorescence by rotating the polarization of all emission photons to the S-plane, the plane for which the diffraction grating is most efficient. Three diffraction gratings are provided supporting wavelength sampling increments of 2.5, 5, and 10 nm. Improvements have been made in the digitization process to increase detection efficiency as well. C1si is calibrated to a high enough standard that it is possible to share and reproduce data between instruments. The algorithm implemented in the EZ-C1 software is able to accurately and repeatedly unmix fluorescent probes with emission peaks separated by as little as 5 nm. It is possible to unmix probes with emission peaks separated by 20 nm with a 10-1 brightness difference. Three probes can be unmixed with emission peaks contained within a 20 nm range. Acquisition is fast enough and the sensitivity is sufficient for C1si to acquire more than 100 frames of spectral time series data without serious photobleaching.     

Frequency-dependent variation in the two-dimensional beam pattern of an echolocating dolphin

Recent recordings of dolphin echolocation using a dense array of hydrophones suggest that the echolocation beam is dynamic and can at times consist of a single dominant peak, while at other times it consists of forward projected primary and secondary peaks with similar energy, partially overlapping in space and frequency bandwidth. The spatial separation of the peaks provides an area in front of the dolphin, where the spectral magnitude slopes drop off quickly for certain frequency bands. This region is potentially used to optimize prey localization by directing the maximum pressure slope of the echolocation beam at the target, rather than the maximum pressure peak. The dolphin was able to steer the beam horizontally to a greater extent than previously described. The complex and dynamic sound field generated by the echolocating dolphin may be due to the use of two sets of phonic lips as sound sources, or an unknown complexity in the sound propagation paths or acoustic properties of the forehead tissues of the dolphin.     

A robust biomarker discovery pipeline for high-performance mass spectrometry data

A high-throughput software pipeline for analyzing high-performance mass spectral data sets has been developed to facilitate rapid and accurate biomarker determination. The software exploits the mass precision and resolution of high-performance instrumentation, bypasses peak-finding steps, and instead uses discrete m/z data points to identify putative biomarkers. The technique is insensitive to peak shape, and works on overlapping and non-Gaussian peaks which can confound peak-finding algorithms. Methods are presented to assess data set quality and the suitability of groups of m/z values that map to peaks as potential biomarkers. The algorithm is demonstrated with serum mass spectra from patients with and without ovarian cancer. Biomarker candidates are identified and ranked by their ability to discriminate between cancer and noncancer conditions. Their discriminating power is tested by classifying unknowns using a simple distance calculation, and a sensitivity of 95.6% and a specificity of 97.1% are obtained. In contrast, the sensitivity of the ovarian cancer blood marker CA125 is approximately 50% for stage I/II and approximately 80% for stage III/IV cancers. While the generalizability of these markers is currently unknown, we have demonstrated the ability of our analytical package to extract biomarker candidates from high-performance mass spectral data.     

Identification of two forms of progesterone receptor from chick oviduct cytosol using non-denaturing gel electrophoresis

Non-denaturing polyacrylamide gel electrophoresis and non-denaturing agarose gel electrophoresis have been used to resolve [3H]R5020-binding components from chick oviduct cytosol. From both gel systems 2 peaks of bound radioactivity are resolved which display these properties of authentic progesterone receptor: binding of R5020: steroid specificity, saturability, and restriction to target tissues. The two peaks are approximately equal in magnitude, and there is no evidence for interconversion of the 2 peaks. The presence or absence of 10-20 mM sodium molybdate during cytosol preparation had no effect on the magnitude or mobility of either peak. Neither peak contains salt-dissociable components which affect its electrophoretic properties, suggesting a possible alteration of native receptor forms during electrophoresis.     

The Spectral Sensitivities of Single Cells in the Median Ocellus of Limulus

The spectral sensitivities of single Limulus median ocellus photoreceptors have been determined from records of receptor potentials obtained using intracellular microelectrodes. One class of receptors, called UV cells (ultraviolet cells), depolarizes to near-UV light and is maximally sensitive at 360 nm; a Dartnall template fits the spectral sensitivity curve. A second class of receptors, called visible cells, depolarizes to visible light; the spectral sensitivity curve is fit by a Dartnall template with λ_max at 530 nm. Dark-adapted UV cells are about 2 log units more sensitive than dark-adapted visible cells. UV cells respond with a small hyperpolarization to visible light and the spectral sensitivity curve for this hyperpolarization peaks at 525–550 nm. Visible cells respond with a small hyperpolarization to UV light, and the spectral sensitivity curve for this response peaks at 350–375 nm. Rarely, a double-peaked (360 and 530 nm) spectral sensitivity curve is obtained; two photopigments are involved, as revealed by chromatic adaptation experiments. Thus there may be a small third class of receptor cells containing two photopigments.     

Synthesis and characterization of hexadecadienyl compounds with a conjugated diene system,sex pheromone of the persimmon fruit moth and related compounds

Hexadecadien-1-ol and the derivatives (acetate and aldehyde) with a conjugated diene system have recently been identified from a pheromone gland extract of the persimmon fruit moth (Stathmopoda masinissa), a pest insect of persimmon fruits distributed in East Asia. The alcohol and acetate showed their base peaks at m/z 79 in a GC-MS analysis by electron impact ionization, but the aldehyde produced a unique base peak at m/z 84, suggesting a 4,6-diene structure. To confirm this inference, four geometrical isomers of each 4,6-hexadecadienyl compound were synthesized by two different routes in which one of two double bonds was furnished in a highly stereoselective manner. Separation of the two isomers synthesized together by each route was facilely accomplished by preparative HPLC. Their mass spectra coincided well with those of natural components, indicating that they were available for use as authentic standards for determining the configuration of the natural pheromone. Furthermore, other hexadecadienyl compounds, including the conjugated diene system between the 3- and 10-positions, were synthesized to accumulate the spectral data of pheromone candidates. 5,7-Hexadecadienal interestingly showed the base peak at m/z 80; meanwhile, the base peaks of its alcohol and acetate were detected at m/z 79 like the corresponding 4,6-dienes. The base peaks of all 6,8-, 7,9-, and 8,10-dienes universally appeared at m/z 67 like 9,11-, 10,12-, and 13,15-dienes, the spectra of which have already been published. Although 3,5-hexadecadienal was not prepared, base peaks at m/z 67 and 79 were recorded for the alcohol and acetate, respectively.     

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections

Summary A component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono-and diformazans are formed, and these exhibit overlapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono-and diformazans in histochemical reactions.     

Fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor

Microbial products formed in biological wastewater treatment systems are closely related to system performance and status, and many of them have fluorescence spectral characteristics. In this work, the fluorescence spectral characteristics of the supernatants from an anaerobic hydrogen-producing bioreactor were studied using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. Since the components of the microbial products are complex, the parallel factor analysis (PARAFAC) method was used to extract the real spectra from the overlapped spectra. Two principal components were identified from the EEM spectra. The peaks at excitation–emission maxima of 280/350 and 350/440 nm were, respectively, attributed to the fluorescence of proteins and NADH. Their real concentrations were quantified using the PARAFAC coupled with the second-order calibration method. Results show that the formation rate of proteins was correlated to the production rate of hydrogen and volatile fatty acids, as well as the substrate degradation rate. A close correlation between the hydrogen partial pressure and the two fluorophores was found out. This study provides a reliable and convenient approach, which could be potentially used for monitoring the wastewater treatment reactor performance through measuring the fluorescence spectra of the supernatant.     

Spectral Sensitivity of the Common Prawn, Palaemonetes vulgaris

The vision of Palaemonetes is of particular interest in view of extensive studies of the responses of its chromatophore systems and eye pigments to light. The spectral sensitivity is here examined under conditions of dark adaptation and adaptation to bright colored lights. In each case the relative number of photons per one-fiftieth sec flash needed to evoke a constant peak amplitude (usually 25 or 50 µv) in the electroretinogram (ERG) was measured at various wavelengths throughout the spectrum. The sensitivity is the reciprocal of this number. In dark-adapted animals the spectral sensitivity curve consists of a broad, almost symmetrical band, maximal at about 540 mµ, with a shoulder near 390 mµ. Adaptation to bright red or blue light, left on continuously throughout the measurements, depresses the 540 mµ peak without notably changing its shape or position, implying that only one visual pigment operates in this region. Adaptation to red light, however, spares a violet-sensitive system, so that a high, narrow peak at 390 mµ now dominates the spectral sensitivity function. The 540 and 390 mµ peaks are apparently associated with different visual pigments; and these seem to be segregated in different receptor systems, since the associated ERG's have markedly different time constants. It is suggested that these two sensitivity bands may represent the red- and violet-sensitive components of an apparatus for color differentiation.     

A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin

The emission spectrum of intact purple membranes of Halobacterium halobium has a very short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components, one of which (component A) corresponds to the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid environment (like the single tryptophan residue in azurin), the other (component I) being due to the emission of buried tryptophan residues located in a rather polar environment. Treatment of bacteriorhodopsin by NaBH4, fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A, an increase in that of component I and the appearance of spectral components corresponding to the emission of surface tryptophan residues. Temperature induces at least two distinct changes of the fluorescence parameters of the protein: one change occurs from 45 to 65 degrees C. the other from 65 to 90 degrees C. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline denaturation of bacteriorhodopsin registered by tryptophan fluorescence begins at pH > 11.0.     

Partitioning plant spectral diversity into alpha and beta components

Plant spectral diversity – how plants differentially interact with solar radiation – is an integrator of plant chemical, structural, and taxonomic diversity that can be remotely sensed. We propose to measure spectral diversity as spectral variance, which allows the partitioning of the spectral diversity of a region, called spectral gamma (γ) diversity, into additive alpha (α; within communities) and beta (β; among communities) components. Our method calculates the contributions of individual bands or spectral features to spectral γ‐, β‐, and α‐diversity, as well as the contributions of individual plant communities to spectral diversity. We present two case studies illustrating how our approach can identify 'hotspots’ of spectral α‐diversity within a region, and discover spectrally unique areas that contribute strongly to β‐diversity. Partitioning spectral diversity and mapping its spatial components has many applications for conservation since high local diversity and distinctiveness in composition are two key criteria used to determine the ecological value of ecosystems.     

Thermoluminescence and electric polarization in chloroplasts

Exposure of pea chloroplasts to electric field causes the appearance of a new thermoluminescence (TL) band at--(40-50) degrees C and a reduction of the intensity of its main bands. Extents of intensity drop are different for different components of TL and depend on the temperature of illumination. The charge traps responsible for the individual TL components seem to be localized in microsurroundings having different field susceptibility. The electric field effects observable at different temperatures are in correlation with the thermodepolarization currents which reflect the mobility and number of charged groups undergoing a field-induced displacement in chloroplast membranes. Dehydration. of chloroplast film preparations causes a reduction in the intensities of the TL peaks and thermodepolarization currents and a shift of the peaks positions toward higher temperatures. It is assumed that the traps of the recombining charges have two different conformations, each with its own frequency factor for the recombination reaction. Changes in the thermoluminescence behavior in applied electric field are due to the polarization of the traps, which increases the existence probability of a conformation with a high frequency factor.     

Development of analytical methods for NMR spectra and application to a 13C toxicology study

Metabolomics offers the potential to assess the effects of toxicants on metabolite levels. To fully realize this potential, a robust analytical workflow for identifying and quantifying treatment-elicited changes in metabolite levels by nuclear magnetic resonance (NMR) spectrometry has been developed that isolates and aligns spectral regions across treatment and vehicle groups to facilitate analytical comparisons. The method excludes noise regions from the resulting reduced spectra, significantly reducing data size. Principal components analysis (PCA) identifies data clusters associated with experimental parameters. Cluster-centroid scores, derived from the principal components that separate treatment from vehicle samples, are used to reconstruct the mean spectral estimates for each treatment and vehicle group. Peak amplitudes are determined by scanning the reconstructed mean spectral estimates. Confidence levels from Mann–Whitney order statistics and amplitude change ratios are used to identify treatment-related changes in peak amplitudes. As a demonstration of the method, analysis of ¹³C NMR data from hepatic lipid extracts of immature, ovariectomized C57BL/6 mice treated with 30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or sesame oil vehicle, sacrificed at 72, 120, or 168 h, identified 152 salient peaks. PCA clustering showed a prominent treatment effect at all three time points studied, and very little difference between time points of treated animals. Phenotypic differences between two animal cohorts were also observed. Based on spectral peak identification, hepatic lipid extracts from treated animals exhibited redistribution of unsaturated fatty acids, cholesterols, and triacylglycerols. This method identified significant changes in peaks without the loss of information associated with spectral binning, increasing the likelihood of identifying treatment-elicited metabolite changes.     

Kinetic multichannel spectroscopy of biological molecules: decomposition of the spectral matrix

The time-resolved difference spectra after flash excitation of various biological molecules are measured on a gated optical multichannel analyzer. Electron transfer between the photoactive covalent label thiouredopyrene-trisulfonate and the heme of cytochrome c and the photocycle of the E204Q mutant bacteriorhodopsin are studied. The spectral matrices containing consecutive difference spectra are analyzed to reveal the reaction kinetics and individual component spectra. Singular value decomposition combined with stoichiometric analysis and self-modeling is demonstrated as a tool for successful dissection of the matrix, despite the difficulties arising from broad, featureless, overlapping spectra and the overlapping kinetics of the components.