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The hyper-Raman scattering (HRS) spectra of biologically significant molecules (D-glucose, L-alanine, L-arabinose, L-tartaric acid) in aqueous solutions are reported. The HRS spectra were measured using a picosecond laser at 532 nm operating at a MHz repetition rate. High signal to noise spectra were collected with a commercial spectrometer and CCD without resonant, nanoparticle, or surface enhancement. The HRS peak frequencies, relative intensities, band assignments, and depolarization ratios are examined. By comparing HRS to Raman scattering (RS) and infrared absorption spectra we verify that the IR-active vibrational modes of the target molecules are observed in HRS spectra but come with substantially different peak intensities. The HRS of the biomolecules as well as water, dimethyl sulfoxide, methanol, and ethanol were deposited into a data repository to support the development of theoretical descriptions of HRS for these molecules. Depositing the spectra in a repository also supports future dual detection RS, HRS microscopes which permit simultaneous high-spatial-resolution vibrational spectroscopy of IR-active and Raman-active vibrational modes.

Hydrogen bonding plays an essential role in biological processes by stabilizing proteins and lipid structures as well as controlling the speed of enzyme catalyzed reactions. Dimethyl sulfoxide-water (DMSO-H 2 O) solution serves as a classical model system by which the direct and indirect effects of hydrogen bonding between water hydrogens and the sulfoxide functional group can be explored. The complex transition from self-bonding to heterogeneous bonding is important, and multiple spectroscopic approaches are needed to provide a detailed assessment of those interactions. In this report, for the first time, hyper-Raman scattering was successfully employed to investigate molecular interactions in DMSO-H 2 O system. We measured the improper blueshift of the C-S and C-H stretching modes of DMSO caused by partial charge transfer and enhanced bond polarization. By detecting differences in the frequency shifts of C-S and C-H modes for low DMSO concentrations (<33 mol%) we find evidence of the intermolecular bonds between water and the DMSO methyl groups. We exploit the high sensitivity of hyper-Raman scattering to the low frequency librations of H 2 O to observe a change in librational mode population providing insight into existing questions about the coordination of H 2 O around DMSO molecules and the formation of the H 2 O shell around DMSO molecules proposed in prior simulation studies. These results demonstrate that hyper-Raman spectroscopy can be a practical spectroscopic technique to study the intermolecular bonding of model systems and test claims about model system bonding generated by theoretical calculations.

Chemical imaging of calcifications was demonstrated in the depth of a tissue. Using long wavelength excitation, broadband coherent anti-Stokes Raman scattering and hierarchical cluster analysis, imaging and chemical analysis were performed 2 mm below the skin level in a model system. Applications to breast cancer diagnostics and imaging are discussed together with the methods to further extend the depth and improve the spatial resolution of chemical imaging.

We introduce a unique technique for generating directional coherent emissions that could be utilized to create coherent sources in a wide range of frequencies from the extreme ultraviolet (XUV) to the deep infrared. This is accomplished without population inversion by pumping a two-level system with a far-detuned strong optical field that induces the splitting of the two-level system. A nonlinear process of four-wave mixing then occurs across the split system, driving coherent emission at sidebands both red- and blue-detuned from the pump frequency, and propagates both forward and backward along the pump beam path. We observed this phenomenon in dense rubidium vapor along both the D 1 and D 2 transitions. The sideband emission exhibits a short pulse duration (<1 ns) with threshold-like behavior dependent on both the pump intensity and Rb vapor density. This technique offers a new capability for manipulating the emission frequency simply through intensity-induced atomic modulation that can be scaled to most frequency regimes using various atomic/molecular ensembles and pump energies., Competing Interests: The authors declare no conflict of interest.

Tunable narrow-band short-pulse coherent emission can be generated by the optical parametric amplification of a seeded continuous wave (CW) laser. However, the residual CW pedestal can affect the accuracy of the optical measurements and the exact interpretation of the experimental data. We demonstrate a simple approach to removing the residual CW seed in a frequency tunable, seeded parametric amplification setup in the nanosecond regime by adding an additional parametric amplification stage which is seeded by an idler wave from the first stage. We validate this method by using a pump-probe experiment in an atomic vapor. Our results show the elimination of an atomic vapor hyperfine pumping signal after the CW pedestal has been removed.

Fast and sensitive Raman spectroscopy measurements are imperative for a large number of applications in biomedical imaging, remote sensing and material characterization. Stimulated Raman spectroscopy offers a substantial improvement in the signal-to-noise ratio but is often limited to a discrete number of wavelengths. In this report, by introducing an electronically-tunable acousto-optical filter as a wavelength selector, a novel approach to a broadband stimulated Raman spectroscopy is demonstrated. The corresponding Raman shift covers the spectral range from 600 cm(-1) to 4500 cm(-1), sufficient for probing most vibrational Raman transitions. We validated the use of the new instrumentation to both coherent anti-Stokes scattering (CARS) and stimulated Raman scattering (SRS) spectroscopies.

A novel concept of Brillouin imaging and sensing via time-resolved optical (BISTRO) measurements is introduced for flow cytometry applications. The system affords robust, maintenance-free and high-speed elasticity-sensitive measurements.

Raman spectroscopy is an important tool in understanding chemical components of various materials. However, the excessive weight and energy consumption of a conventional CCD-based Raman spectrometer forbids its applications under extreme conditions, including unmanned aircraft vehicles (UAVs) and Mars/Moon rovers. In this article, we present a highly sensitive, shot-noise-limited, and ruggedized Raman signal acquisition using a time-correlated photon-counting system. Compared with conventional Raman spectrometers, over 95% weight, 65% energy consumption, and 70% cost could be removed through this design. This technique allows space- and UAV-based Raman spectrometers to robustly perform hyperspectral Raman acquisitions without excessive energy consumption.

Fast and sensitive Raman spectroscopy measurements are imperative for a large number of applications in biomedical imaging, remote sensing and material characterization. In this report, by introducing an electronically-tunable acousto-optical filter as a wavelength selector, we demonstrated a novel instrumentation to the broadband coherent Raman spectroscopy. System's tunability allows assessing Raman transitions ranging from <400 cm(-1) to 4500 cm(-1). We validated the use of the new instrumentation by collecting coherent anti-Stokes spectra and stimulated Raman spectra of various samples.

Random lasers are a developing class of light sources that utilize a highly disordered gain medium as opposed to a conventional optical cavity. Although traditional random lasers often have a relatively broad emission spectrum, a random laser that utilizes vibration transitions via Raman scattering allows for an extremely narrow bandwidth, on the order of 10 cm(-1). Here we demonstrate the first experimental evidence of lasing via a Raman interaction in a bulk three-dimensional random medium, with conversion efficiencies on the order of a few percent. Furthermore, Monte Carlo simulations are used to study the complex spatial and temporal dynamics of nonlinear processes in turbid media. In addition to providing a large signal, characteristic of the Raman medium, the random Raman laser offers us an entirely new tool for studying the dynamics of gain in a turbid medium.

Subsurface analysis of chemical species is imperative for biomedical diagnostics and imaging, homeland security, and pharmaceutical and other industries; however, the access to the object of interest is often obscured by an optically scattering medium which limits the ability to inspect the chemical composition of the sample. In this report, we employ coherent Raman microspectroscopy in a combination with a hierarchical cluster analysis to mitigate the effect of scattering and demonstrate the identification of multiple chemical species.

We experimentally investigated the nonlinear optical interaction between the instantaneous four-wave mixing and the cascaded quadratic frequency conversion in commonly used nonlinear optical KTP and LiNbO3 with the aim of a possible background suppression of the non-resonant background in coherent anti-Stokes Raman scattering. The possibility of background-free heterodyne coherent anti-Stokes Raman scattering microspectroscopy is investigated at the interface formed by a liquid (isopropyl alcohol) and a nonlinear crystal (LiNbO3).

Stimulated Raman scattering (SRS) microscopy is a powerful tool for chemically-sensitive non-invasive optical imaging. However, ultrafast laser sources, which are currently employed, are still expensive and require substantial maintenance to provide temporal overlap and spectral tuning. SRS imaging, which utilizes continuous-wave laser sources, has a major advantage, as it eliminates the cell damage due to exposure to the high-intensity light radiation, while substantially reducing the cost and complexity of the set-up. As a proof-of-principle, we demonstrate microscopic imaging of dimethyl sulfoxide using two independent, commonly used lasers, a diode-pumped, intracavity doubled 532-nm laser and a He-Ne laser operating at 632.8-nm.

We propose and experimentally validate a technique for four-wave mixing background suppression in coherent anti-Stokes Raman spectroscopy. It is based on the interaction of the signals generated from the Kerr third-order nonlinearity and the cascaded quadratic process in a nonlinear crystal. Theoretical analysis agrees well with the experimental results, which provide a quantitative assessment of different contributions and allow extraction of the nonlinearity parameters.

High dynamic range optical-to-near-infrared transmission measurements for different parts of human body in the spectral range from 650 to 950 nm have been performed. Experimentally measured spectra are correlated with Monte Carlo simulations using chromaticity coordinates in CIE 1976 L*a*b* color space. Both a qualitative and a quantitative agreement have been found, paving a new way of characterizing human tissues in vivo. The newly developed experimental and computational platform for assessing tissue transmission spectra is anticipated to have a considerable impact on identifying favorable conditions for laser surgery and optical diagnostics, while providing supplementary information about tissue properties.

In this letter, we, for the first time, report on coherent anti-Stokes Raman scattering (CARS) spectroscopy of an ensemble of silicon nanowires (SiNWs) formed by wet chemical etching of crystalline silicon with a mask of silver nanoparticles. The fabricated SiNWs have diameter ranged from 30 to 200 nm and demonstrate both visible and infrared photolumine cence (PL) and spontaneous Raman signal, with their intensities depending on presence of silver nanoparticles in SiNWs. The efficiency of CARS in SiNW ensembles is found to be significantly higher than that in crystalline silicon. The results of CARS and PL measurements are explained in terms of resonant excitation of the electron states attributed to silicon nanoparticles.

In this report, we show the collection of spatial information through a turbid medium by coherent Raman microspectroscopic imaging. In particular, the technique is capable of identifying anthrax endospores inside a sealed paper envelope.

A powerful combination of chemically specific Raman excitation and deep tissue ultrasound imaging holds the promise to attain spatially resolved distribution of chemical compounds inside the scattering medium. In this report, an attempt is made to evaluate the recent achievements and possible challenges with an eye on potential clinical applications.

Nonlinear Raman microspectroscopy based on a broadband coherent anti-Stokes Raman scattering is an emerging technique for noninvasive, chemically specific, microscopic analysis of tissues and large population of cells and particles. The sensitivity of this imaging is a critical aspect of a number of the proposed biomedical application. It is shown that the incident laser power is the major parameter controlling this sensitivity. By careful optimizing the laser system, the high-quality vibrational spectra acquisition at the multi-kHz rate becomes feasible.

Coherent Raman microspectroscopy imaging is an emerging technique for noninvasive, chemically specific optical imaging, which can be potentially used to analyze the chemical composition and its distribution in biological tissues. In this report, a hierarchical cluster analysis was applied to hyperspectral coherent anti-Stokes Raman imaging of different chemical species through a turbid medium. It was demonstrated that by using readily available commercial software (Cytospec, Inc.) and cluster analysis, distinct chemical species can be imaged and identified through a rather thick layer of scattering medium. Once the clusters of different chemical composition were distinguished, a phase retrieval algorithm was used to convert coherent anti-Stokes Raman spectra to Raman spectra, which were used for chemical identification of hidden microscopic objects. In particular, applications to remote optical sensing of potential biological threats and to imaging through a layer of skin tissue were successfully demonstrated.

Osteoporosis is a bone disease characterized by reduced mineral content with resulting changes in bone architecture, which in turn increases the risk of bone fracture. Raman spectroscopy has an intrinsic sensitivity to the chemical content of the bone, but its application to study bones in vivo is limited due to strong optical scattering in tissue. It has been proposed that Raman excitation with photoacoustic detection can successfully address the problem of chemically specific imaging in deep tissue. In this report, the principal possibility of photoacoustic imaging for detecting mineral content is evaluated.

We propose and experimentally demonstrate a new coherent anti-Stokes Raman scattering setting, which relies on a coherent excitation of Raman vibration using a broadband ultrashort laser pulse and signal read-out using a conventional continuous wave laser radiation. Such an exotic arrangement does not require any synchronization of two laser sources and can be used for direct comparison of amplitudes of nonlinear and spontaneous Raman signals.

Purpose: The purpose of this work is to demonstrate that higher amplitude of ultrashort laser induced photoacoustic signal can be achieved by multiple-pulse excitation when the temporal duration of the pulse train is less than the minimum of the medium's thermal relaxation time and stress relaxation time. Thus, improved signal-to-noise ratio can thus be attained through multiple-pulse excitation while minimizing the energy of each pulse., Methods: The authors used a Michelson interferometer together with a picoseconds laser system to introduce two 6 ps pulses separated by a controllable delay by introducing a path length difference between the two arms of the interferometer. The authors then employed a series of three interferometers to create a pulse train consisting of eight pulses. The average pulse energy was 11 nJ and the temporal span of the pulse train was less than 1 ns., Results: The detected peak-to-peak amplitude of the multiple-pulse induced photoacoustic waves were linearly dependent on the number of pulses in the pulse train and such a linearity held for different optical absorption coefficients. The signal-to-noise ratio improved when the number of pulses increased. Moreover, nonlinear effects were not detected and no photoacoustic saturation effect was observed., Conclusions: The authors have shown that multiple-pulse excitation improves the signal-to-noise ratio through an accumulated energy deposition effect. This method is invaluable for photoacoustic measurements that require ultrashort laser pulses with minimized pulse energy to avoid laser damage.

Stimulated Raman scattering as a promising way of expanding the tunability of ultrafast lasers and as an exciting new biomedical imaging modality capable of selective excitation and chemically-specific diagnostics of molecular species.

Nonlinear Raman scattering is an emerging spectroscopy technique for non-invasive microscopic imaging. It can produce a fluorescence background free vibrational spectrum from a microscopic volume of a sample providing chemically specific information about its molecular composition. We analyze the ability of nonlinear Raman microspectroscopy to detect low concentrated molecular species and evaluate its applicability to study complex solutions.

Single bacterial spores were analyzed by using nonlinear Raman microspectroscopy based on coherent anti-Stokes Raman scattering (CARS). The Raman spectra were retrieved from CARS spectra and found to be in excellent agreement with conventionally collected Raman spectra. The phase retrieval method based on maximum entropy model revealed significant robustness to external noise. The direct comparison of signal amplitudes exhibited a factor of 100 stronger CARS signal, as compared with the Raman signal.

The output of a long-cavity 2 MHz all-solid-state Nd:YVO(4) picosecond oscillator with nonlinear optical mirror mode locking is used for direct pumping of an optical parametric amplifier seeded with white-light continuum generated in optical fiber by the same laser to generate tunable (5-12 microm) mid-IR radiation.

Anisotropic photonic crystal structures consisting of birefringent porous silicon layers with alternating porosity were fabricated. The in-plane birefringence formed as a result of anisotropic etching in Si(110) results in unique multilayered structures with two distinct photonic bandgaps for orthogonal light polarizations. Nonlinear optical studies based on the third-harmonic generation from these structures demonstrate variation in the symmetry of the nonlinear optical response.

Most of the known methods to measure the nonlinear optical properties of materials deal with the bulk properties, but there are many demanding applications that require those measurements to be done on a single particle or a single molecule. We report a novel application of nonlinear optics to measure the third-order nonlinear optical susceptibility of nanoparticles in solutions. By measuring the power of the third harmonic generated in a diluted solution of nanoparticles, both the size and chi(3) can be extracted from a simple set of measurements.

We report an efficient red-shifted continuum generation of picosecond pulses in conventional optical fibers. By using a novel highrepetition rate, high-energy oscillator operating at the fundamental wavelength of 1064 nm, we achieved more than 60% of the output energy in the spectral range from 1150 to 1300 nm, perfectly suitable for broadband coherent anti-Stokes Raman spectroscopy.

Third-harmonic microscopy is one of the emerging techniques for noninvasive microscopic imaging of biological structures. We use a novel technique for nonlinear optical material characterization and study the effect of different environment and the structural sensitivity of the third harmonic. In particular, a transformation of collagen in solution is observed for the first time using third-harmonic generation. We also study the ultimate limits of the third harmonic to detect micro- and nanoscopic features inside living cells and find that structures as small as 50 nm can be detected using the current level of technology.

Intensity-dependent polarization rotation that results from the simultaneous action of two third-order processes, self-phase modulation and four-wave mixing, is observed. The effect is recorded along the fourfold axis of a YVO(4) crystal. The angle of rotation is proportional to the square of the input intensity-crystal length product. This is to the authors' knowledge the first experimental evidence showing that intensity-dependent polarization rotation can be related to the real part of cubic susceptibility.

The nonlinear optical properties of solid-solid phase transformation in vanadium dioxide are studied. It is found that the efficiency of the third-harmonic optical signal generated from the surface of the material increases by 1.5 orders of magnitude as a function of this phase transformation. Microscopy studies show the hysteresis of the phase transformation on a micrometer-size scale.

A new cubic nonlinear optical effect in which a linearly polarized wave propagating in a single quadratic medium is converted into a wave that is cross polarized to the input wave is observed in BBO crystal. The effect is explained by cascading of two different second-order processes: second-harmonic generation and difference frequency mixing.

A method for disintegration of cells of group A streptococcus on the Edebeau extrusion press was developed. The level of disintegration was controlled by cell count in stained preparations, Coultier electronic counter and electron microscope. The streptococcus cell disintegration in the Edebeau extrusion press at a temperature of --40 degrees, a pressure of 3200 kg/cm2 and two cycles of the process was completed by 96-98%.

Precipitation by ammonium sulfate and a subsequent purification of the culture fluid of Actinomyces levoris by gel-filtration through Sephadex G-25, ion-exchange chromatography on DEAE-cellulose and CM-cellulose resulted in an enzyme which activley lyzes the cell walls of a hemolytic streptococcus of group A. The molecular weight (12,500), isoelectric point (pI 10,6) and amino acid composition of the enzyme were determined. The enzyme specificity was assayed using peptidoglycane isolated rom the cell walls of streptococcus of group A used as a substrate. An analysis of the hydrolysis products of peptidoglycane showed that the enzyme under study is an endo-beta-N-acetylmuramidase (EC 3.2.1.17).

As revealed, autolytic enzymatic system of hemolytic streptococcus, group A, expressed the maximal activity at the end of the exponential growth phase. Autolysis was accompanied by the release of N-acetylaminosugars and of free amino group. Optimal conditions for the lytic system function were chosen (composition, ionic strength, and buffer pH). The activating action of trypsin and chemotrypsin was demonstrated.

Endo-N-acetylglucoseaminidase (EC 3.2.1.30) was prepared from the cultural broth of Streptomyces levoris 96 using precipitation with ammonium sulfate, gel filtration on Sephadex G-25 and ion exchange chromatography on DEAE-cellulose. The isoelectric point of the enzyme is 4.2 Str. levoris produces various quantities of the enzyme depending on the composition of the growth medium. A medium in which the enzyme is produced in maximal amounts has been selected. The effect of peptidoglycan, cell walls and chitin added to the medium on the enzyme biosynthesis was studied. Chitin induced biosynthesis of the enzyme by 35--45%.

The content of protein and carbohydrate polymers was estimated in the cell wall of Streptococcus, group A, type 29. A method was developed for analysing peptidoglycane in a polysaccharide-peptidoglycane complex after the prior oxidation by sodium periodate. It was found that the cell wall peptidoglycane bears two carbohydrate and three amino acid residues, i. e. N-acetylglucosamin, muramic acid, glutamic acid, alanine and lysine, in the ratio 1:1:1:4:1, respectively. The data on the cell wall composition prior to and after its oxidation with sodium periodate are given, and the ratio between the main structural components is determined: proteins (60% mol), polysaccharide (23% mol), peptidoglycane (17% mol).

Muramidase which actively lyses the cell walls of group A hemolytic streptococci has been isolated from the culture fluid of Actinomyces levoris by precipitation on ammonium sulfate, gel filtration on Sephadex G-25, ion exchange chromatography on DEAE cellulose and double electrofocusing. The enzyme thus obtained has shown its maximum activity at 40-50 degrees C. At 60 degrees C muramidase was completely inactivated. The enzyme has a wide range of optimum pH values: 5.0-9.5. The highest percentage of lysis of streptococcal cell walls has been observed in plycine-NaOH and potassium phosphate buffers at pH 8.0. Muramidase completely lysed Streptococcus pyogenes cells of different serological groups except enterococci (group D) and staphylococci. This sign allows to differentiate group D streptococcus from streptococci of other groups.

A method of determining aminosaccharides (muramic acid, glucosamine and galactosamine) by means of a carbohydrate analyzer "Biotronic" using the cation-exchange resin DC-6 A ("Durrum") was developed. Chromatographic conditions correspond to the conditions of neutral sugar analysis on a column with DCh-4 resin, that enables after a slight modification of the analyzer to pass from the determining of aminosaccharides to the determining of neutral sugars. The method was used for determining the carbohydrate composition of streptococcus cell walls. The results obtained allow to conclude that using this method one can get more information on the hydrocarbon composition of various biological objects than using the method of aminosaccharide determining by means of aminoacid anylyzer, which is widely in practice nowadays.

More than 80 cultures of actinomycetes belonging to different taxanomic groups were studied with a purpose of screening actinomycetes actively producing enzymes lyzing the cell walls of group A streptococci. 31 strains of the actinomycetes producing enzymes which lyzed the cell walls by 20-50 and 60-80 per cent within 1 and 4 hours respectively were selected. The proteolytic activity of the enzymes produced by these strains was also studied. It was shown that 4 cultures, i.e. Actinomyces albus, strains 6 and 9, Actinomyces levoris, strain 29 and Actinomyces gibsonii, strain 42 were of interest as organisms producing enzymes which lyzed the streptococcal cell wall without impairing its antigenic components.

The composition of the enzyme complex produced by Actinomyces levoris which exhibits lytic action on the cell walls of Streptococcus lactis was studied by gel filtration on Sephadex G-25, ion exchange chromatography and isoelectric focusing. The complex was found to contain at least seven enzymes having different isoelectric points and substrate specificity. The enzymes were divided into three groups. (1) The enzyme with pI 10.2 seems to be a specific glucosidase; it possesses a high activity of the cell wall lysis and lacks a proteolytic activity. (2) The enzymes with pI 9.2 and 9.0 are, apparently, specific lytic proteases; they display both lytic and proteolytic activities. (3) The enzymes with pI 10.0, 9.5, 5.7 and 4.2 are, presumably, non-specific lytic proteases; they have a low lytic activity and a high proteolytic activity.

The effect of Streptomyces griseus pronase on Streptococcus group A cell walls was studied. Cell walls were shown to be lysed by pronase, the lysis level being dependent on the molarity of the potassium-phosphate buffer used. With an increase in the buffer molarity from 0.005 M to 0.05 M lysis of cell walls decreased from 70-80% to 30%. By DEAE-cellulose chromatography lysates were separated into two fractions the first of which contained a group specific polysaccharide. A preparative method of obtaining a group specific polysaccharide of Streptococcus group A using Streptomyces griseus pronase under mild conditions is described.

A comparative study of lipoteichoic acid preparations extracted from Streptococcus pyogenes with cold and hot phenol and trichloracetic acid was made. The most delicate way for isolation of lipoteichoic acid from the given microorganisms is cold phenol extraction.

Ribosomes from Streptococcus pyogenes, group A, strain 29 were studied. A comparison of different methods of ribosomal isolations has shown that the homogenous ribosomal samples can be obtained by the method of differential ultracentrifugation using tris-HCl buffer. The ribosomes of S. pyogenes had the sedimentation coefficient of 70S and consisted of 65% of protein and 35% of nucleic acids; the ribosomes dissociated into subparticles with the sedimentation coefficients of 50S and 30S under a low magnesium concentration. Thus the S. pyogenes ribosomes do not differ from the ribosomes of procaryotes. It was shown that the ratios of 70S, 50S and 30S ribosomal subparticles in the cells depend on the growth phase of S. pyogenes. The cells of the middle and the late logarithmic phase contained 50S and 30S particles in a stoichiometric ratio. In the cells of the late stationary growth phase there was a deficiency of 30S ribosomal subparticles which does not result from a loss during the isolation procedure, as it was already observed in the initial 30S fraction.