Quantitative correlative proton and electron microprobe analysis of biological specimens

To investigate the possibility of quantitative correlative proton microprobe (PMP) and electron microprobe (EMP) analysis of biological soft tissue, model specimens were analyzed by both techniques. The specimens consisted of freeze-dried sections of gelatin containing known concentrations of nickel chloride. Both for PMP and for EMP, the signal was expressed as the ratio of the characteristic intensity and the continuum intensity in a peak-free region of the spectrum. With both techniques, calibration curves (signal versus known concentration) obtained, showed a deviation from linearity at high nickel concentrations. However, a linear relation (correlation coefficient 0.996) was obtained in a plot of EMP signal versus PMP signal. This indicates that quantitative correlative PMP and EMP analysis can be carried out by using the same standard for both analytical techniques.     

Quantitative analysis of knock-on and thermal damage of biological specimens by electron irradiation in transmission electron microscopy

High-energy electrons are able to transfer momentum to nuclei, which results in displacement on to the interstitial lattice sites with a maximum transferred energy of 4 · 10⁴ eV for carbon at 100 keV. Moreover, most of the energy dissipated in energy losses is converted into heat, which results in melting and evaporation.The specimen temperature rise was calculated by the heat conduction theory and confirmed by the specimen drift due to the thermal damage. The damage can be reduced by a small area of illumination, the use of a metal-coated microgrid and small area scanning.A further displacement due to the knock-on collision and the resulting etching rate of biological specimens was measured. The damage is proportional to the current density in c cm^-2 at the specimen. The allowable maximum dose was obtained from the measurement of an etching rate with the weight loss and dry density of the specimens.It was found that the images affected by the electron irradiation, in which -H, C-H, C-N bonds break molecules in proteinaceous biological specimens are removed, and the remaining molecules are changed to stable carbon-rich molecules by deposition, polymerization and contamination. In addition, defect images were observed in high contrast, when compared with unaffected images taken with a small area scanning method.     

A review of sources of spurious silicon peaks in electron microprobe X-ray spectra of biological specimens.

The presence of Si peaks in X-ray spectra of biological specimens can lead to misinter-pretation of data if one is unaware of potential sourees of artifactual Si. Silicontcontamination may be acquired from oils or greases during the course of specimen preparation or from the vacuum system of the electron microscope. In energy dispersive X-ray detection, the level of the Si peak cannot be reduced below that of the internal fluorescence peak obtained from the dead layer of the detector.     

Quantitative analysis of sputtering due to ion beam bombardment of solids and biological specimens in high resolution electron microscopy

When a positively charged ion beam is used to bombard a solid target, most of the atoms are displaced and sputtered according to the atomic sputtering theory. In the case of biological specimens, most of the bond-breaking molecules in proteins are removed, when based on the molecular sputtering theory. It was found that the thinning rate for solids and the etching rate for biological specimens, when prepared by a normal double fixation and staining method, can be measured from the sputtering yield and density of the specimens. It was also found that the thinning and etching rates depend on the removal weight per sublimation energy and bonding energy, respectively. The angular distribution of sputtering yield, its dependence on incident angle and the secondary electron emission yield were measured, and the optimum etching condition of the incidence was obtained. Experiments showed that the in situ observation of intracellular structures of biological specimens prepared by ion beam etching can be a very effective method in electron microscopy.     

High-performance electron tomography of complex biological specimens

We have evaluated reconstruction methods using smooth basis functions in the electron tomography of complex biological specimens. In particular, we have investigated series expansion methods, with special emphasis on parallel computation. Among the methods investigated, the component averaging techniques have proven to be most efficient and have generally shown fast convergence rates. The use of smooth basis functions provides the reconstruction algorithms with an implicit regularization mechanism, very appropriate for noisy conditions. Furthermore, we have applied high-performance computing (HPC) techniques to address the computational requirements demanded by the reconstruction of large volumes. One of the standard techniques in parallel computing, domain decomposition, has yielded an effective computational algorithm which hides the latencies due to interprocessor communication. We present comparisons with weighted back-projection (WBP), one of the standard reconstruction methods in the areas of computational demand and reconstruction quality under noisy conditions. These techniques yield better results, according to objective measures of quality, than the weighted backprojection techniques after a very few iterations. As a consequence, the combination of efficient iterative algorithms and HPC techniques has proven to be well suited to the reconstruction of large biological specimens in electron tomography, yielding solutions in reasonable computation times.     

Electron microscopy,electron diffraction,and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Electron microscopy, electron diffraction, and element analysis of wet biological specimens

Temperature controlled differentially pumped environmental chambers now allow more routine examination of wet specimens in the electron microscope. A sensitive test of their efficiency is the ability to provide high resolution electron diffraction patterns from wet, unfixed protein microcrystals. Fortunately, wet specimens can be prepared with only a few tens of nanometers thickness of remaining water, so extraneous electron scattering by liquid water can be kept to a minimum. It still remains to be determined whether microprobe analysis (X-ray or electron energy-loss spectroscopy) using wet specimens gives better element localization in cells than the current freezing methods. More extensive comparisons are also required of the ultrastructural preservation and visibility of macromolecules immersed in a thin layer of water vs immersion in a thin layer of amorphous ice. However, the recent introduction of commercial forms of the necessary equipment now make these comparisons more feasible.     

Quantitative X-ray microanalysis of halogen elements in biological specimens

Summary Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.     

Quantitative X-ray microanalysis of halogen elements in biological specimens

Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.     

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory.

X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV.

From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.     

Devices facilitating the preparation of biological specimens for electron microscopy

The construction of a matrix is described which facilitates the process of placing biological objects into polymer media in order to prepare ultrathin sections without the employment of gelatin, starch and polyethylene capsules that can be used only once. The construction of a reactor for cytochemical assays is presented. The apparatus can be used to locate enzymes within the cell, and to identify microorganisms. A modification of the dialysis technique for microbiological objects is proposed which accelerates and simplifies the process.     

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy

Summary The ability of tannic acid to enhance binding of glycosaminoglycans to purified collagen was analysed in an in vitro system using amino sugar analysis on an amino acid analyser, transmission electron microscopy, and scanning electron microscopy. Collagen was purified by digestion with trypsin, papain, and hyaluronidase. Purified collagen was incubated with hyaluronic acid or with chondroitin sulphate glycosaminoglycan and then treated with tannic acid. Tannic acid was found to enhance retention during preparation for electron microscopy of either of the glycosaminoglycans onto collagen fibres. The ability of tannic acid to enhance binding of collagen and glycosaminoglycans might explain, at least in part, its structural reinforcement effect on resected synovial joint-apposing surfaces during preparation for scanning electron microscopy.     

Quantitative gas-liquid chromatographic determination of ftorafur and 5-fluorouracil in biological specimens

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.     

Compressed sensing for electron cryotomography and high-resolution subtomogram averaging of biological specimens

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Distribution of ions inNeurospora crassa determined by quantitative electron microprobe analysis

Summary It was investigated whether the low membrane potential at the tip of hyphae ofNeurospora crassa could be described as a diffusion potential. The distribution of Na, K and P inNeurospora was measured by electron microprobe analysis of freeze-dried hyphae. Quantitative determinations were made with freeze-dried gelatin standards and with crystalline standards. The local concentrations of Na and K were in accordance with the theory that at the tip of the hyphe, these ions are distributed passively across the plasma membrane.     

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Application of a proton microprobe to biomedical research

The capability of PIXE analysis to simultaneously detect trace elements with Z≥14, with a high power of detection, can be exploited in biomedical research if the diameter of the proton beam is reduced to micrometer dimensions. In this case, trace analyses of small particles or small parts of a larger specimen are rendered possible without deteriorating the detection limits of PIXE. The measurements yield a completely new type of information on the biological microstructure. In order to fully utilize the abilities of the combined method, however, sample preparation techniques, and irradiation procedures have to be adapted to each analysis problem. Examples of application of the Bochum Proton Microprobe will be used to demonstrate how and to what extent this can be achieved for different types of biomedical problems.