Beam shaping and probe characterization in the scanning electron microscope.

Here we demonstrate the use of nanofabricated grating holograms to diffract and shape electrons in a scanning electron microscope. The diffraction grating is placed in an aperture in the column. The entire diffraction pattern can be passed through the objective lens and projected onto the specimen, or an intermediate aperture can be used to select particular diffracted beams. We discuss several techniques for characterizing the diffraction pattern. The grating designs can incorporate features that can influence the phase and intensity of the diffracted SEM probe. We demonstrate this by producing electron vortex beams.

Imaging of tissue sections with very slow electrons.

The examination of thin sections of tissues with electron microscopes is an indispensable tool. Being composed of light elements, samples of living matter illuminated with electrons at the usual high energies of tens or even hundreds of kiloelectronvolts provide very low image contrasts in transmission or scanning transmission electron microscopes. Therefore, heavy metal salts are added to the specimen during preparation procedures (post-fixation with osmium tetroxide or staining). However, these procedures can modify or obscure the ultrastructural details of cells. Here we show that the energy of electrons used for the scanned transmission imaging of tissue sections can be reduced to mere hundreds or even tens of electronvolts and can produce extremely high contrast even for samples free of any metal salts. We found that when biasing a sufficiently thin tissue section sample to a high negative potential in a scanning transmission electron microscope, thereby reducing the energy of the electrons landing on the sample, and collecting the transmitted electrons with a grounded detector, we obtain a high contrast revealing structure details not enhanced by heavy atoms. Moreover, bombardment with slow electrons sensitively depolymerises the resin in which the tissue is embedded, thereby enhancing the transmitted signal with no observable loss of structure details. The use of low-energy electrons requires ultrathin sections of a thickness of less than 10nm, but their preparation is now possible. Ultralow energy STEM provides a tool enabling the observation of very thin biological samples without any staining. This method should also be advantageous for examination of 2D crystals, thin films of polymers, polymer blends, etc.

Very low energy electron microscopy of graphene flakes.

Commercially available graphene samples are examined by Raman spectroscopy and very low energy scanning transmission electron microscopy. Limited lateral resolution of Raman spectroscopy may produce a Raman spectrum corresponding to a single graphene layer even for flakes that can be identified by very low energy electron microscopy as an aggregate of smaller flakes of various thicknesses. In addition to diagnostics of graphene samples at larger dimensions, their electron transmittance can also be measured at very low energies.

Collection of secondary electrons in scanning electron microscopes.

Collection of the secondary electrons in the scanning electron microscope was simulated and the results have been experimentally verified for two types of the objective lens and three detection systems. The aberration coefficients of both objective lenses as well as maximum axial magnetic fields in the specimen region are presented. Compared are a standard side-attached secondary electron detector, in which only weak electrostatic and nearly no magnetic field influence the signal trajectories in the specimen vicinity, and the side-attached (lower) and upper detectors in an immersion system with weak electrostatic but strong magnetic field penetrating towards the specimen. The collection efficiency was calculated for all three detection systems and several working distances. The ability of detectors to attract secondary electron trajectories for various initial azimuthal and polar angles was calculated, too. According to expectations, the lower detector of an immersion system collects no secondary electrons I and II emitted from the specimen and only backscattered electrons and secondary electrons III form the final image. The upper detector of the immersion system exhibits nearly 100% collection efficiency decreasing, however, with the working distance, but the topographical contrast is regrettably suppressed in its image. The collection efficiency of the standard detector is low for short working distances but increases with the same, preserving strong topographical contrast.

Single electron response of the scintillator-light guide-photomultiplier detector.

The time response of a scintillator-light guide-photomultiplier combination was measured with a time-constant of 3 ns. Single detected electrons were recognizable at the output of the photomultiplier. The distribution of the number of photoelectrons produced by one detected electron and the pulse-height distribution of the photomultiplier output pulses were analysed. Statistical noise computed from these distributions was compared with the noise produced by the dark current of the photomultiplier.

New multichannel electron energy analyzer with cylindrically symmetrical electrostatic field.

This article discusses an electron energy analyzer with a cylindrically symmetrical electrostatic field, designed for rapid Auger analysis. The device was designed and built. The best parameters of the analyzer were estimated and then experimentally verified.

Corrections of magnification and focusing in a cathode lens-equipped scanning electron microscope.

One of the well-proven and efficient methods of obtaining a very low-energy impact of primary electrons in the scanning electron microscope is to introduce a retarding field element below the pole piece of the objective lens (OL). It is advantageous to use the specimen alone as the negatively biased electrode (i.e., cathode of the cathode lens). The optical power of the cathode lens modifies some of the standard parameters of the image formation such as relation of working distance to OL excitation or magnification to the scanning coils current, the impact angle of primary electrons, and so forth. In computer-controlled electron microscopes these parameters, particularly with regard to focusing and magnification, can be corrected automatically. Derivation of algorithms for such corrections and their experimental verifications are presented in this paper. Furthermore, a more accurate analytical expression for the focal length of an aperture lens is derived.

Non-cytolytic inhibition of hepatitis B virus replication in human hepatocytes.

BACKGROUND/AIMS: Interferon-gamma (IFN-gamma) has been shown to abolish hepatitis B virus (HBV) gene expression and replication in HBV transgenic mice without destroying infected hepatocytes. We investigated the characteristics of IFN-gamma induced non-cytolytic inhibition of viral replication in human HBV infection. METHODS: We used an in vitro model where lymphocytes from 15 HBsAg positive patients were co-cultured with transfected hepatocytes supporting HBV replication. The effector and target cells were separated by a membrane, which allowed transfer of soluble factors only, to determine whether IFN-gamma produced from antigen-specific CD4+ T cells or mitogen stimulated lymphocytes inhibits HBV replication. RESULTS: IFN-gamma produced following lymphocyte stimulation reduced cytoplasmic HBV DNA in the target cells. The degree of HBV DNA reduction correlated with the level of IFN-gamma in the supernatants. Further investigations using naturally infected human hepatocytes confirmed that recombinant IFN-gamma reduces HBV DNA and HBV RNA in these cells as well, in parallel with the induction of cellular interferon-responsive genes. This antiviral effect was without significant cytotoxicity and was more pronounced in hepatocytes from patients with low HBV replication. CONCLUSIONS: These results provide direct evidence that IFN-gamma can inhibit both HBV transcription and replication in human hepatocytes without cell lysis.

Scanning electron microscopy of nonconductive specimens at critical energies in a cathode lens system.

A method for scanning electron microscopy imaging of nonconductive specimens, based on measurement and utilisation of a critical energy, is described in detail together with examples of its application. The critical energy, at which the total electron yield curve crosses the unit level, is estimated on the basis of measurement of the image signal development from the beginning of irradiation. This approach, concentrated onto the detected signal as the only quantity crucial for the given purpose of acquiring a noncharged micrograph, evades consequences of any changes in an irradiated specimen that influence the total electron yield curve and possibly also the critical energy value. Implementation of the automated method, realised using a cathode lens-equipped scanning electron microsope (SEM), enables one to establish a mean rate of charging over the field of view and its dependence on the electron landing energy. This dependence enables one to determine the energy of a minimum damage of the image of the given field of view. Factors influencing reliability and applicability of the method are discussed and examples of noncharged micrographs of specimens from both life and material science fields are presented.

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.

A novel in-lens detector for electrostatic scanning LEEM mini-column

A novel principle of an in-lens detector of very slow electrons is described and the detector efficiency discussed. The detector was built into a coaxial column for a Cylindrical Mirror Analyser for Auger electron microanalysis. In order to obtain a very low energy scanned imaging, a cathode lens was formed between the final electrode of the column and a negatively biased specimen. The signal electrons accelerated within the cathode lens field enter the column and after being mirrored back impact a micro-channel-plate based detector fitted around the optical axis. The acceptance of the detector, expressed as a ratio of the number of electrons impacting the detector to the full emission of a cosine source, was calculated to be 0.86 for 1 eV and 0.985 for 10 eV electrons. Then, the efficiency of conversion into output pulses is 0.35 and 0.31, respectively; these parameters are superior to those of conventional SEM detectors for secondary electrons. Micrographs taken at low energies ranging down to units of eV are presented.

[Chemotherapy of resistant and recurrent lymphoma based on a combination of ifosfamide and etoposide. Antitumor effects, toxicity and stimulation of peripheral stem cells]. / Chemoterapie rezistentních a relabujících lymfomu zalozená na kombinaci ifosfamidu a etopozidu. Protinádorový efekt, toxicita a stimulace periferních kmenových bunek.

BACKGROUND: Treatment of Hodgkin's disease (HD) and non-Hodgkin lymphomas (NHL) is still unsatisfactory in patients resistant to primary therapy or those with early relapses. The objective of the trial was to test whether salvage regimens based on a combination of iphosphamide and etopozide have a sufficient anti-lymphoma effect, while the toxicity is still acceptable, and in conjunction with growth factors as satisfactory mobilizing potential for the collection of peripheral stem cells (PBSC). METHODS AND RESULTS: A group of 40 patients with relapsing and/or primary therapy resisting lymphomas (14 NHL, 26 HD) were treated by life saving or stimulating chemotherapy VIM (etopozide, iphosphamide, methotrexate) and MINE (iphosphamide mitoxanthrone, etopozide; mostly NHL). If the response to these regimes was inadequate, to some patients in addition mini (dexa) regimes were administered, BEAM and DHAP resp., with the objective to achieve maximum reduction of the tumourous mass before high-dosage treatment with the support of autologous PBSC. The authors administered 119 cycles of salvage chemotherapy (51 VIM, 46 MINE, 22 mini-dexa-BEAM and DHAP). The toxicity of chemotherapy and the therapeutic response were evaluated according to WHO criteria. The toxicity of VIM and MINE, with the exception for mobilization of desirable transient leukopenia, was not serious. During stimulation of PBSC on average three leukophereses were made and on average 9.9. 10(6) CD34+ cells/kg body weight of the patient were obtained and 53.2. 10(4) CFU-GM/kg (VIM), and 13.5. 10(6) CD34+ cells/kg 53.4. 10(4) CFU-GM/kg (MINE) resp. A total of 64% (MINE) and 61% (VIM) therapeutic responses were obtained, 14% (MINE) and 26% (VIM) complete remissions and 50% (MINE) and 35% (VIM) partial remissions. CONCLUSIONS: Life saving regimes, VIM and MINE, have a good antilymphoma activity, low toxicity and in combination with growth factors (filgrastim) they ensure a good collection of PBSC. As compared with other regimens, in particular for stimulation, VIM and MINE appear to be better.

Nasopharyngeal characteristics in children with cleft lip and palate.

Cephalometric assessment of the nasopharynx and its adjacent structures was carried out in two experimental groups of 5-year-old male patients with unilateral cleft lip and palate. The first group included individuals who had not had surgery, and the second was comprised of individuals who were at least 1 year postpalatoplasty with primary pharyngeal flap. They were compared with a control group of boys of comparable average age who did not have clefts. Both groups of patients with clefts showed a reduction of the nasopharyngeal bony framework related to the posterior position and decreased posterior height of the maxilla without hypertrophy of the adenoids. The smaller nasopharyngeal airway was consistent with the reduction of the size of the bony nasopharynx. There was shortening of the anterior cranial base located in the region of the middle cranial fossa. The height of the body of the sphenoid bone was reduced, but the angle of the cranial base was within normal limits.