Alexander K. Petrov

Individual Escherichia coli Cells Studied from Light Scattering with the Scanning Flow Cytometer

BACKGROUNDnFlow cytometry is a powerful tool for the analysis of individual particles in a flow. Differential light scattering (an indicatrix) was used for many years to obtain morphologic information about microorganisms. The indicatrices play the same role for individual particle recognition as a spectrum for substance characterization. We combined two techniques to analyze the indicatrix of the cells for the purpose of developing a database of light-scattering functions of cells.nnnMETHODSnThe scanning flow cytometer (SFC) allows the measurement of the entire indicatrix of individual particles at polar angles ranging from 5 degrees to 100 degrees. In this work, light-scattering properties of Escherichia coli have been studied both experimentally and theoretically with the SFC and the T-matrix method, respectively. The T-matrix method was used because of the nonspherical shape of E. coli cells, which were modeled by a prolate spheroid.nnnRESULTSnThe indicatrices of E. coli cells were stimulated with T-matrix method at polar angles ranging from 10 degrees to 60 degrees. The absolute cross-section of light scattering of E. coli has been determined comparing the cross section of polystyrene particles modeled by a homogeneous sphere. The E. coli indicatrices were compared for logarithmic and stationary phases of cell growth.nnnCONCLUSIONSnThe indicatrices of E. coli were reproducible and could be used for identification of these cells in biologic suspensions. The angular location of the indicatrix minimum can be used in separation of cells in logarithmic and stationary phases. To use effectively the indicatrices for that purpose, the light-scattering properties of other microorganisms have to be studied.

Calibration-free method to determine the size and hemoglobin concentration of individual red blood cells from light scattering

At present, hemoglobin concentration and the volume of an erythrocyte can be determined from the intensities of light scattered by an individual cell at fixed angular intervals. This method is used in modern hemoglobin analyzers, but it requires calibration of optical and electronic units by certified particles of known size and refractive index. We describe a method that is based on the parametric solution of an inverse light-scattering problem and does not require a calibration procedure. The method is based on the use of parameters of the entire angular light-scattering pattern, called an indicatrix here. These parameters do not depend on the absolute intensity of light scattering. The indicatrix parameters form approximating equations that relate these parameters to the size and the phase-shift parameters of the particle. The applicability of the method is demonstrated by measurement of the indicatrices of individual sphered erythrocytes. The indicatrices of the individual erythrocytes were measured with a scanning flow cytometer at an angular range of from 15 to 55 deg. The volume and the hemoglobin concentration have been calculated by use of the developed method and by fitting of the experimental indicatrices to the indicatrices calculated from the Mie theory.

Poly(vinyl amine)-silica composite nanoparticles: models of the silicic acid cytoplasmic pool and as a silica precursor for composite materials formation.

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the soluble silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.

Soft Ablation of Biological Objects Caused by Free-Electron Laser Submillimeter Radiation

In 1919, Aston [1] built the first mass spectrometer, which was used to determine the isotopic composition of many chemical elements. For example, the stable isotopes 13 C, 15 N, 17 O, and 2 H(D) have been discovered. Later, chemists mastered this method. At present, a mass spectrum can be recorded for any substance that can be transferred into a gas phase, the molecular weight being determined from the mother peak of the spectrum, and the molecular structure being reconstructed from the fragments of molecules [2]. The use of mass spectrometry in biology was prevented not so much by the large weights of biological molecules as by the impossibility to transfer them into a gas phase. This obstacle was overcome when the MALDI method [3] had been developed. In this method, a matrix containing protein is exposed to an intense laser impulse 10 –9 –10 –6 s in length. This causes ablation, i.e., knocks molecules and their fragments out of the surface of the substrate. Usually, standard UV or near-IR lasers with a high quantum energy are used, which causes substantial destruction of molecules of the sample analyzed.

Dissection of the frustules of the diatom Synedra acus under the action of picosecond impulses of submillimeter laser irradiation

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30–100xa0ps) but high-power (1xa0MW) terahertz laser impulses at 5.6xa0MHz frequency.

Mild ablation of biological objects under the submillimeter radiation of the free electron laser

First line of FEL with smooth tuning of irradiation wavelength 100-200 microns and 400 W power is commissioned in Siberian Center for Photochemical Research. Irradiation was used for mild ablation of DNA, enzymes, and proteins. Transfer from surface occurs without molecular destruction. Particle size was determined using aerosol diffusion spectrometer.

Infrared Multiple Photon Dissociation of Chloromethyltrifluorosilane

Infrared multiphoton absorption and dissociation of chloromethyltrifluorosilane molecules under the action of pulsed transversely excited atmospheric pressure CO2 laser were experimentally studied. Dissociation products were analyzed. The dissociation proceeds via chlorine atom transfer from carbon to silicone. High degrees of silicon isotope separation were achieved. The presence of α‐chlorine atom in a silicon organic compound brings about a significant improvement in multiple photon dissociation characteristics and an essential increase in isotopic selectivity.

Status of FEL-SUT project, and the experimental setup for multiphoton dissociation and isotope separation in the gaseous phase

Abstract The IR FEL Research Center of the Science University of Tokyo (FEL-SUT) is open for users to develop new applications of IR FEL in a wide field of material science, chemical technology and bio-chemical applications. The FEL is based on 35xa0MeV linac operated at the frequency of 2856xa0MHz (s-band). The FEL covers the wavelength range from 5 to 16xa0μm with the micropulse duration of 1–2xa0ps, macropulse duration of 1xa0μs, macropulse repetition rate of 10xa0Hz and the overall average power of 1xa0W. We report the present status of the Center and an experimental setup designed and constructed for the experiments on multiphoton dissociation and isotope separation.

15N isotope-selective infrared multiphoton dissociation of nitromethane by a free electron laser

Successful experiments on the isotope-selective infrared multiphoton dissociation (IR MPD) of nitromethane molecules in the region of stretching vibrations of the NO2 group have been performed for the first time under IR free electron laser (FEL) irradiation in a mixture with the natural content of the15N isotope of 0.4%. The content of the15N isotope in the products of NO dissociation varies within 0.1–1.6% as a function of the laser radiation frequency.

FEL THz irradiation approach for the biochip production standardization

The terahertz emission was applied to the development of the technology for the biochip production standardization. The approach is based on the method of soft nondestructive ablation developed by authors, which means the transfer of target DNA to aerosol phase from solid substrate of a biochip under action of the terahertz emission.