植物叶片的相位解析光声光谱和光声相位谱

用相位解析法获得的未损伤植物叶片叶肉组织的光声光谱,与叶片的光声相位谱进行了比较．发现植物叶片的光声相位谱与叶绿体光吸收带之间呈现互补关系．叶片表皮的光声相位谱存在持有的波谷结构．不同调制频率的光声相位谱有较大的差异．这些现象表明光声相位谱与光声光谱一样,也可对未损伤的层状生物样品作深度剖面分析．     

Photoacoustic spectroscopy of Anacystis nidulans. II. Characterization of pigment holochroms and thermal deactivation spectrum

The photoacoustic spectrum of Anacystis nidulans recorded at room temperature is qualitatively similar to low-temperature absorption or fluorescence excitation spectra. The bands of pigment holochroms are well resolved compared to room-temperature absorption spectra. The thermal deactivation spectrum obtained by extrapolating acoustic data for an infinitely thin sample indicates that the photosynthetic efficiency decreases from phycocyanin to chlorophyll a and carotenoids.     

Photoacoustic spectra of needles as an indicator of the activity of the photosynthetic apparatus of healthy and damaged conifers

Photoacoustic spectra were measured at chopping frequencies of the excitation light at 22 and 515 Hz in order to detect differences between needles from healthy and damaged conifers ( Abies alba Mill, and Picea abies Karst.) with respect to pigment distribution and possibly also to photosynthetic activity. Fully green needles of healthy trees exhibit photoacoustic spectra at 22 Hz with a maximum in the red absorption region of the chlorophyll. This maximum is lost with increasing damage to needles and chlorophyll breakdown. The photoacoustic spectra at 22 Hz of the damaged needles therefore are characterized by a higher signal in the blue-light region as compared to that in the red-light region. This can be quantified by forming the ratio of the photoacoustic signals at 675 and 475 nm (ratio PA 675/475). The needles of the damaged trees possess a lower photosynthetic activity, as seen from the CO₂ fixation rate and the variable fluorescence (Rfd-values). It is assumed that the changes in the PA-spectra of the needles from damaged trees are the result of this decline of the photosynthetic apparatus and its function. In contrast to the PA-spectrum at 22 Hz the PA-spectra at 515 Hz, where the PA-signals primarily emanate from the epidermal layer, exhibit a different shape with a maximum near 550 nm. In the needles from the damaged trees the PA-signals are higher, particularly in this maximum range of the spectrum, than those of fully green needles from healthy trees.     

Application of Photoacoustic Phase Angle Spectroscopy (φAS) to Eumelanins and Pheomelanins

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment. Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.     

Measurement of protein biomass by Fourier transform infrared-photoacoustic spectroscopy

A relatively new analytical technique, Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS), provides spectra of bacteria, fungi, and other microorganisms in solid states not suitable for conventional absorption spectroscopy. In this paper the feasibility of quantitative measurement of protein biomass on solid substrates by FTIR-PAS is examined and discussed. By measuring photoacoustic absorption bands from amide groups in the protein of microorganisms, the increase in biomass that occurs during growth was monitored directly and accurately. Incorporation of polyacrylonitrile into the sample as an internal standard was shown to be a convenient method for improving both the reliability and the range of detection by photoacoustic spectroscopy. Results of FTIR-PAS measurements of known quantities of microbial mass in simulated growth experiments suggest that the technique may be especially suitable for assays of microorganisms used in solid-state biosyntheses of drugs, hormones, and other biological agents.     

Simultaneous detection of photosynthetic energy storage and oxygen evolution in leaves by photothermal radiometry and photoacoustics

We have measured simultaneously the photothermal radiometry and the photoacoustic signals from intact leaves. We have confirmed that while the former senses that part of the modulated absorbed radiation not used in photosynthesis, but converted into heat, the latter, at low modulation frequencies, senses not only this heat but also the modulated oxygen evolution resulting from photosynthesis in the leaf. When photosynthetic activity is saturated upon additional excitation with strong non-modulated light, the photothermal radiometry signal increases (virtually all absorbed modulated light being converted into heat), while at the same time the photoacoustic signal decreases, because virtually no modulated oxygen evolution occurs any more. At higher modulation frequencies the behaviour of the photoacoustic signal closely follows that of the photothermal radiometry signal. We have used combined photothermal radiometry / photoacoustic measurements to estimate directly the yield of chemical energy storage in various plant species which applies for different times after excitation. Measurement of light saturation curves for wheat and Siberian pea bush leaves and of action spectra for the latter confirm the similarity between photothermal radiometry and high-frequency photoacoustic signals, and their difference from the low-frequency photoacoustic signal. Combined use of photothermal radiometry (or high-frequency photoacoustics) and low-frequency photoacoustics can thus provide more information than any one method alone. Experiments on intact chloroplasts and on a blue-green alga demonstrate that photothermal radiometry and photoacoustic methodologies can also be used for these tissues.     

Two-dimensional mass spectra generated from the analysis of 15N-labeled and unlabeled peptides for efficient protein identification and de novo peptide sequencing

Protein identification has been greatly facilitated by database searches against protein sequences derived from product ion spectra of peptides. This approach is primarily based on the use of fragment ion mass information contained in a MS/MS spectrum. Unambiguous protein identification from a spectrum with low sequence coverage or poor spectral quality can be a major challenge. We present a two-dimensional (2D) mass spectrometric method in which the numbers of nitrogen atoms in the molecular ion and the fragment ions are used to provide additional discriminating power for much improved protein identification and de novo peptide sequencing. The nitrogen number is determined by analyzing the mass difference of corresponding peak pairs in overlaid spectra of (15)N-labeled and unlabeled peptides. These peptides are produced by enzymatic or chemical cleavage of proteins from cells grown in (15)N-enriched and normal media, respectively. It is demonstrated that, using 2D information, i.e., m/z and its associated nitrogen number, this method can, not only confirm protein identification results generated by MS/MS database searching, but also identify peptides that are not possible to identify by database searching alone. Examples are presented of analyzing Escherichia coli K12 extracts that yielded relatively poor MS/MS spectra, presumably from the digests of low abundance proteins, which can still give positive protein identification using this method. Additionally, this 2D MS method can facilitate spectral interpretation for de novo peptide sequencing and identification of posttranslational or other chemical modifications. We envision that this method should be particularly useful for proteome expression profiling of organelles or cells that can be grown in (15)N-enriched media.     

In situ optical spectroscopy of some systems of biological interest

Monitoring the optical absorption or emission spectrum of a condensed phase sample offers information about the supramolecular assembly, packing effects and other features characteristic of the phase that would be missed when one studies solution-state spectra. We have used the technique of photoacoustic spectroscopy to study intact biological specimens, such as algae, parasite cells and the eye lens. Such a study has offered information about the status of endogenous hemin inPlasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to doin situ fluorescence spectroscopy on isolated intact eye lenses, which has enabled us to follow the photochemistry and the status of the photoproduct of the oxidation of the trp residues of the crystallins of the lens.     

Photothermal spectroscopy of bacteriochlorophyll-lipoprotein complexes.

Photoacoustic spectra at room and 85 K temperatures as well as photothermal beam deflection spectra of bacteriochlorophyll-lipoprotein complexes from purple bacterium Chromatium minutissimum were measured. Spectra were compared and obtained differences were tentatively explained by various inertion of these two methods. Photothermal beam deflection method measure the heat which is generated in close surroundings of absorbing pigment molecule, whereas usage of more inert photoacoustic signal is averaged over contributions from various pigments located in a larger sample volume and therefore is similar to absorption spectra.     

Probing Red Blood Cell Morphology Using High-Frequency Photoacoustics

A method that can rapidly quantify variations in the morphology of single red blood cells (RBCs) using light and sound is presented. When irradiated with a laser pulse, an RBC absorbs the optical energy and emits an ultrasonic pressure wave called a photoacoustic wave. The power spectrum of the resulting photoacoustic wave contains distinctive features that can be used to identify the RBC size and morphology. When particles 5–10 μm in diameter (such as RBCs) are probed with high-frequency photoacoustics, unique periodically varying minima and maxima occur throughout the photoacoustic signal power spectrum at frequencies >100 MHz. The location and distance between spectral minima scale with the size and morphology of the RBC; these shifts can be used to quantify small changes in the morphology of RBCs. Morphological deviations from the normal biconcave RBC shape are commonly associated with disease or infection. Using a single wide-bandwidth transducer sensitive to frequencies between 100 and 500 MHz, we were able to differentiate healthy RBCs from irregularly shaped RBCs (such as echinocytes, spherocytes, and swollen RBCs) with high confidence using a sample size of just 21 RBCs. As each measurement takes only seconds, these methods could eventually be translated to an automated device for rapid characterization of RBC morphology and deployed in a clinical setting to help diagnose RBC pathology.     

Comparison of solid and solution state protein structures. Photoacoustic study of solid state bovine methemoglobin derivatives

In an effort to examine the consequences of phase state changes on protein structures, optical absorption spectra of several bovine hemoglobin derivatives in lyophilized, ammonium sulfate-precipitated, and crystalline states were examined. Absorption spectra were used to compare protein conformations. Differences between solution and several solid state spectra were apparent. They were greatest for lyophilized preparations, were inhibited by ligand binding, the tightest binding ligand being the most effective inhibitor, and were substantially or totally reversible upon dissolving the solid state protein. Though the magnitude of spectral changes varied, they all indicated characteristically different proportions of the protein were converted to a reversible hemichrome in each type of solid state preparation. The extent of hemichrome formation was correlated with the degree to which the protein tends to be dehydrated in each solid state preparation. This may reflect the role of water in determining the three-dimensional structure of hemoglobin. Absorption spectra of solid state hemoglobin preparations were obtained from photoacoustic spectra using a novel method for spectral analysis. Results presented here demonstrate the utility of this procedure in probing structures of solid state proteins.     

Excitation Energy Transfer in Cyanobacteria Synechocystis Embedded in Polymer Film and Partially Bleached by Polarized Irradiation

The polarized absorption, photoacoustic, fluorescence emission, and fluorescence excitation spectra of whole cells of cyanobacteria Synechocystis sp. embedded in a polymer film were measured. The bacteria cells, as it follows from anisotropy of absorption and fluorescence spectra, were even in a non-stretched polyvinyl alcohol film oriented to a certain extent. The measurements were done for such film in order to avoid the deformation of cyanobacteria shapes. Part of the samples was bleached by irradiation with strong polarized radiation with electric vector parallel to the orientation axis of cells. The anisotropy of photoacoustic spectra was higher than that of absorption spectra and it was stronger changed by the irradiation. Polarized fluorescence was excited in four wavelength regions characterised by different contribution to absorption from various bacteria pigments. The shapes of emission spectra were different depending on wavelength of excitation, polarization of radiation, and previous irradiation of the sample. The fluorescence spectra were analysed on Gaussian components belonging to various forms of pigments from photosystems (PS) 1 and 2. The results inform about excitation energy transfer between pools of pigments, differently oriented in the cells. Energy of photons absorbed by phycobilisomes was transferred predominantly to the chlorophyll of PS2, whereas photons absorbed by carotenoids to chlorophylls of PS1.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

Quantitative measurement of two-component pH-sensitive colorimetric spectra using multilayer neural networks

The purpose of this research was to develop a noise tolerant and faster processing approach for in vivo and in vitro spectrophotometric applications where distorted spectra are difficult to interpret quantitatively. A PC based multilayer neural network with a sigmoid activation function and a generalized delta learning rule was trained with a two component (protonated and unprotonated form) pH-dependent spectrum generated from microspectrophotometry of the vital dye neutral red (NR). The network makes use of the digitized absorption spectrum between 375 and 675 nm. The number of nodes in the input layer was determined by the required resolution. The number of output nodes determined the step size of the quantization value used to distinguish the input spectra (i.e. defined the number of distinct output steps). Mathematic analysis provided the conditions for which this network is guaranteed to converge. Simulation results showed that features of the input spectrum were successfully identified and stored in the weight matrix of the input and hidden layers. After convergent training with typical spectra, a calibration curve was constructed to interpret the output layer activity and therefore, predict interpolated pH values of unknown spectra. With its built-in redundant presentation, this approach needed no preprocessing procedures (baseline correction or intensive signal averaging) normally used in multicomponent analyses. The identification of unknown spectra with the activities of the output layer is a one step process using the convergent weight matrix. After learning from examples, real time applications can be accomplished without solving multiple linear equations as in the multiple linear regression method. This method can be generalized to pattern oriented sensory information processing and multi-sensor data fusion for quantitative measurement purposes.     

Circular dichroism of helical polynucleotides calculated by the linear response method

Linear response theory in the decorrelation or random-phase approximation is used to calculate the absorption and CD spectra of model helical polymers, including single-stranded polyadenylic acid. The method, which makes use of infinite polymer selection rules for the linear response tensor, has the advantages that (1) only a few three-dimensional matrices need be inverted; (2) spectral band shapes of the polymer arise naturally from those of the monomer, as well as from the geometry-dependent interactions in the helix; and (3) the spectral dependence on geometrical factors of the helix is made transparent. It is found that the structure of the polymer CD spectrum depends critically on monomer bandshape. An asymmetric CD spectrum, similar to some experimental spectra, arises from either a Gaussian or a composite monomer band. Single-stranded polyadenylic acid spectra are sensitive to helix geometry in the region 200–240 nm, in reasonable agreement with experimental spectra. This sensitivity arises from the 207-nm monomer transition, and the results suggest that this region of the spectrum should be more fully exploited as a tool for helix geometry studies.     

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.     

PDN-1: a spin-labeled analog of cis-diamminedichloroplatinum(II). Biophysical studies in aqueous, lipophilic, and biological media

Biophysical and biochemical studies on a biradical-labeled analog of Cisplatin, PDN-1, in aqueous, biological, and lipophilic media are presented. The ESR spectra from PDN-1 in aqueous meida show five-line spectra typical of biradicals in which intramolecular spin exchange occurs. The spectrum of PDN-1 in 1-octanol is characterized by three broad lines overlying a fourth line that is approximately three times as wide as the others. Although the characteristics of the aqueous spectra differ substantially from those of the octanol spectra, both the midfield peak height and the integral of the absorption spectrum can be used as a linear measure of PDN-1 concentration in either media. The smallest concentration used in these linear calibrations was equivalent to the detection of 60 femptomoles of PDN-1 at a signal-to-noise ratio of 30. PDN-1 is shown to "decay" in tissue culture medium which involves either the reduction of a nitroxyl moiety, the labilization of an amino bond, or a combination of both. Spectra of intracellular PDN-1 show that both a mobile ("free") fraction and slowly tumbling ("bound") fraction of the compound can be detected within CHO cells. The spin-labeled cisplatin method is at least as sensitive as conventional chromatographic and spectroscopic methods, yet has the added advantage of offering information about the molecular environment of the complex.     

Controlling Optical Absorption of Graphene in Near-infrared Region by Surface Plasmons

Nowadays, graphene has many applications in optical instruments, biosensors, gas sensors, photovoltaic cells, and so on. In this study, we aimed at investigating the optical properties of graphene under the influence of plasmons created in one-dimensional photonic crystal structure by making use of the absorption spectrum. We put the gold photonic crystal in adjacent to graphene and placed an antireflection layer on top of it. Then, we studied the behavior of graphene absorption peaks in a near-infrared region. By analyzing the graphene behavior in this region, we observed that graphene absorption was increased up to 40% and graphene absorption value in absorption peak, absorption peak wavelength, absorption spectra width, and also its absorption spectra in a wide wavelength range from 1000 to 2500 nm, could be controlled by making use of different factors such as the substance of antireflection layer and photonic crystal geometric dimensions. This structure can make many applications possible for graphene such as using it to build biosensors to identify uric acid and some of the lipids that have specific significances in detecting atherosclerotic lesions as well as diagnosing the states of disease.     

Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins.