We have fabricated nanometer spherical silicon and germanium particles by dissolving semiconductors in some melted metals (aluminum, indium). The first transmission electron microscopy images and electron microdiffraction patterns of the spherical Si and Ge particles are presented. The obtained spherical particles have sizes from hundreds nanometers to tens angstroms, the diameters of the smallest ones are about 1.5 nm that are close to calculated sizes of hypothetic silicon "fullerenes" Si60.
A new versatile near-field scanning optical microscope (NSOM) for technical and biological application have been developed in our laboratory. The microscope can be operated in all known modes, viz., scanning optical, photon tunneling and apertureless photon tunneling modes. Different methods of NSOM tip production are discussed in the paper. The maximal displacement of the coarse adjustment unit is 1 mm the approach step is about 0,05 μm. The NSOM scanner consist of a mechanical scanner with large displacement range (1 mm) on which is mounted a fine piezoscanner with a scanning range about 4 μm. The maximal scanning range of the NSOM is about 1 mm laterally and 4 μm in perpendicular to sample surface direction with rough selection of area for study in the total area about 15x15 mm under conventional optical microscopy observation (maximal magnification 100). As a sample holder are used either a glass prism in photon tunneling mode or a flat glass in scanning optical mode. The microscope has a digital control unit connected with IBM PC through a parallel interface. The NSOM high rigidity makes it possible to operated in ordinary laboratory without any additional vibration isolation devices.
KEYWORDS: 3D image processing, Bacteria, Atomic force microscopy, Microscopy, Visualization, Photomicroscopy, Transmission electron microscopy, Picosecond phenomena, 3D visualizations, Scanning electron microscopy
The vast majority of information about cells and cell organelle structure were obtained by means of transmission electron microscopy investigation of cells serial thin sections. However often it is very difficult to derive information about 3D structure of specimens from such electron micrographs. A new program restoring 3D image of cells from the serial thin sections micrographs have been developed in our lab. The program makes it possible to visualize a 3D image of cell and obtain image of inner cell structure in arbitrary plane. The plague bacteria and anthrax cells with spore were visualized with resolution about 70 nm by means of the program.
The software supporting SPM is a complex which includes a control device and data processing parts. The device control part often depends on physical realization of the microscope. A new program which is developing in our lab is not connected to a definite scanning device. It has a common interface to all devices. That makes possible the interaction with any scanning microscope even non-existed yet. Software also includes easy comprehensible user interface, algorithms library, database, internet connection and import/export modules.
Transmission electron microscopy investigation of thin sections remains the major method of cells inner structure study with high resolution. However, the present-day technique of cells preparation make it impossible to study a number of biological samples, such as very small quantity of lymph cells of little insects. A new technique of cells preparation has been developed in our lab, which allows to obtain a thin sections of ultra small quantity of cells. Structure of lymph cells of flea was investigated by the technique.
Scanning optical far and near-field microscopes resolution is limited by light spot size used to scan a sample. The focusing elements in scanning microscopes are ordinary used lenses with dimensions of rather more than a light wavelength. The focus spot size of such lens is limited by well-known diffraction limit. However, if a lens dimension is comparable with a light wavelength the Fresnel's diffraction formula of focus spot size calculation is not suitable. At present, there are no data about theoretical or experimental studies of the small lenses and even about their fabrication methods. A new fabrication method of small spherical glass lenses with diameters from 1 micrometers to 100 micrometers is considered, and some theoretical approaches to the focus spot size calculation and its experimental measurement are discussed in the paper. The lenses fabrication method is based on melting of fine glass powder with oxygen-acetylene burner with subsequent lenses selection under conventional light microscope. To calculate a spot size of the small lens the Mie theory of light scattering in near field by small spherical particles has been used. The calculated light intensity diagrams for spherical lens in near field are presented.
A scanning optical far and near-field microscopes resolution is limited by focused light spot size used to scan a sample. As a focusing element in scanning microscopes are ordinary used lenses with dimensions of rather more than a light wavelength. The focus spot size of such lens is limited by well-known diffraction limit. However, if a lens dimension is comparable to a light wavelength the Fresnel's diffraction formula of focus spot size calculation is not suitable. At present, there are no data about theoretical or experimental studies of the small lenses and even about the ones fabrication methods. This paper is considers the fabrication method of small spherical glass lenses with diameters from 1 um to 100 um and discusses some theoretical approaches to the focus spot size calculation and it experimental measurement. The lenses fabrication method is based on melting of fine glass powder with oxygen-acetylene burner with subsequent lenses selection under conventional light microscope. To calculate a spot size of the small lens the Mie theory of light scattering in near field by small spherical particles has been used. The light intensity diagrams of sphere lens of various diameters calculated by developed program are presented.
Numerical simulation of image formation in near field optical microscopy is needed to understand a relationship between near field images and actual structure of sample since the image can be differs strongly from real structure. In order to estimate the near field image formation, two different approaches are used, namely numerical solution of integral equations with boundary element method or moment method and analytical solution of light scattering problem obtained by assumption of some hypotheses. The first approach results in reasonably complicated algorithm of calculation and often causes a bed conditioned system of linear equations to be formed. The approach based on an analytical solution of scattering problem is more preferable since allow to reduce the algorithm and increase the calculations speed. A new program of simulation of near field image formation for arbitrary sample shape has been developed. The program is based on the results of analytical calculation of near field light diffraction on sinusoidal diffraction grating of arbitrary corrugation amplitude. The sample shape is presented as a number of sinusoidal gratings of various amplitudes by Fourier transformation, for each of the gratings the light scattering amplitude is calculated and the amplitudes are summarized.
KEYWORDS: 3D image processing, Bacteria, Visualization, Microscopy, Transmission electron microscopy, Photomicroscopy, Image visualization, Scanning electron microscopy, Atomic force microscopy, Electron microscopy
The vast majority information about cells and cell organellae structure were obtained by means of transmission electron microscopy investigation of cells serial thin sections. However often it is very difficult to derive information about 3-D structure of specimen from such electron micrographs. A new program restoring 3-D image of cells from the serial thin sections micrographs have been developed in our lab. The program makes it possible to visualize a 3-D image of cell and obtain image of inner cell structure in arbitrary plane. The plague bacteria and anthrax cells with spore were visualized with resolution about 70 nm by means of the program.
Transmission electron microscopy investigation of thin sections remains the major method of cells inner structure study with high resolution. However, the present-day technique of cells preparation make it impossible to study a number of biological samples, such as very small quantity of lymph cells of little insects (spiders, fleas, etc.). A new technique of cells preparation has been developed in our lab, which allow to obtain a thin sections of ultra small quantity of cells (less then 100). Structure of lymph cells of flea was investigated by the technique.
To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.
As a branch of scanning probe microscopy, near field scanning optical microscopy (NSOM) has been the most intensively developed for the past few years. A wide variety of types of NSOM have been created of recent years. However, a versatile NSOM that allow to investigate a same with different near field microscopy is not worked out up to now. In our lab a new versatile NSOM for biological application have been developed. The microscope can be operated in all known modes, viz., scanning optical, photon tunneling and aperture less photon tunneling modes. The basic element of the NSOM design is a coarse adjustment unit on which various scanners and sample holders are mounted. The maximal displacement of the coarse adjustment unit is 1 mm the approach step is about 50 micrometers . The NSOM scanner consists of a mechanical scanner with large displacement range on which is mounted a fine piezoscanner with t a scanning range about 2 micrometers . The maximal scanning range of the NSOM is about 1 mm laterally and 4 micrometers in perpendicular to sample surface direction with rough selection of area for study in the total are about 3 X 3 mm under conventional optical microscopy observation. As a sample holder are used either a glass prism in photon tunneling mode or a flat glass in scanning optical mode. The microscope has a digital control unit connected with IBM PC through a parallel interface. Different biological samples were investigated by means of the NSOM with resolution better than 15 nm. The NSOM high rigidity makes it possible to operate in ordinary laboratory without any additional vibration isolation devices.
A fine structure of bacteria flagella is an important problem of molecular cell biology. Bacteria flagella are the self-assembled structures that allow to use the flagellum protein in a number of biotechnological applications. However, at present, there is a little information about high resolution scanning probe microscopy study of flagellum structure, in particular, about investigation of Vibrio cholerae flagella. In our lab have been carried out the high resolution comparative investigation of V. cholerae flagella by means of various microscopes: tunneling (STM), scanning force (SFM) and electron transmission. As a scanning probe microscope is used designed in our lab versatile SPM with replaceable measuring heads. Bacteria were grown, fixed and treated according to the conventional techniques. For STM investigations samples were covered with Pt/Ir thin films by rotated vacuum evaporation, in SFM investigations were used uncovered samples. Electron microscopy of the negatively stained bacteria was used as a test procedure.
In our biophysical laboratory a new scanned probe microscope (SPM) for technical and biological application has been developed. The SPM allows to investigate sample surface by means of three different near field microscopes: scanning tunneling microscope, atomic force microscope and near field scanning optical microscope. The SPM is very rigid and can be operated in ordinary laboratory without any vibration isolation. The scanning area of the microscope is about 10 X 10 micrometers . Different technical and biological applications of the SPM are demonstrated. Results of the SPM investigations of different carbon, metal and dielectric films are described. The SPM comparison study of electrical breakdown and the conduction bistable switching effect in thin dielectric films of oxides and fluorides of some rare earth metals has been discussed. Some biological applications of the SPM viz. visualization of different bacteria (E.Coli, plague, cholera, staphylococcus), bacteria thin sections, macromolecules (plague proteins) and plague phage has been described.
Microscopy investigations of S-layers of plague microbes have been carried out. A protein forming S-layer was isolated, purified and its biochemical properties were studied. Images of plague cells with and without S-layer and images of isolated S-layer protein arrays on solid supports were obtained by transmission electron microscopy. An isolated protein forming the layer is self-assembled into crystalline structure with hexagonal pores are of regular size about 4 - 8 nm, which was observed by transmission electron micrographs. The STM images of plague microbes with S-layer and without one and the STM and AFM images of isolated S-layer protein arrays on mica surface were obtained.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.